American Journal of Botany 98(12)

American Journal of Botany 98(12): 2049–2063. 2011.
PHYLOGENETIC AND POPULATION GENETIC ANALYSES OF
DIPLOID LEUCAENA (LEGUMINOSAE; MIMOSOIDEAE) REVEAL
CRYPTIC SPECIES DIVERSITY AND PATTERNS OF DIVERGENT
ALLOPATRIC SPECIATION1
Rajanikanth Govindarajulu2,5, Colin E. Hughes3,4, and C. Donovan Bailey2,4
2Department
3Institute
of Biology, P. O. Box 30001 MSC 3AF, New Mexico State University, Las Cruces, New Mexico 88001 USA;
of Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland; and 4Department of Plant
Sciences, South Parks Road, University of Oxford, Oxford OX13RB UK
• Premise of the study: Leucaena comprises 17 diploid species, five tetraploid species, and a complex series of hybrids whose
evolutionary histories have been influenced by human seed translocation, cultivation, and subsequent spontaneous hybridization. Here we investigated patterns of evolutionary divergence among diploid Leucaena through comprehensively sampled
multilocus phylogenetic and population genetic approaches to address species delimitation, interspecific relationships, hybridization, and the predominant mode of speciation among diploids.
• Methods: Parsimony- and maximum-likelihood-based phylogenetic approaches were applied to 59 accessions sequenced for
six SCAR-based nuclear loci, nrDNA ITS, and four cpDNA regions. Population genetic comparisons included 1215 AFLP loci
representing 42 populations and 424 individuals.
• Results: Phylogenetic results provided a well-resolved hypothesis of divergent species relationships, recovering previously
recognized clades of diploids as well as newly resolved relationships. Phylogenetic and population genetic assessments identified two cryptic species that are consistent with geography and morphology.
• Conclusions: Findings from this study highlight the importance and utility of multilocus data in the recovery of complex evolutionary histories. The results are consistent with allopatric divergence representing the predominant mode of speciation
among diploid Leucaena. These findings contrast with the potential hybrid origin of several tetraploid species and highlight the
importance of human translocation of seed to the origin of these tetraploids. The recognition of one previously unrecognized
species (L. cruziana) and the elevation of another taxon (L. collinsii subsp. zacapana) to specific status (L. zacapana) is consistent with a growing number of newly diagnosed species from neotropical seasonally dry forests, suggesting these communities harbor greater species diversity than previously recognized.
Key words: allopatric speciation; cryptic species; Leguminosae; Leucaena; Leucaena cruziana; Leucaena zacapana; phylogeny; population genetics.
Patterns of diversification among species can be explained
by a wide variety of evolutionary mechanisms. Geographic isolation leading to divergence among populations is generally
considered to be the most common mode of speciation (e.g.,
Grant, 1971), but reticulate evolution and polyploidy following
hybridization between divergent populations can also prompt
sudden reproductive isolation and speciation in plants (e.g.,
Rieseberg, 1997; Mavárez et al., 2006). Hybridization is well
1 Manuscript
received 3 June 2011; revision accepted 12 October 2011.
Earlier fieldwork that laid the foundations and provided much of the
material for this research benefitted from the support of numerous
colleagues in Mexico and notably, J. L. Contreras, H. Ochoterena, M.
Sousa, and S. Zárate, as well as ongoing support from the Instituto de
Biología of the Universidad Nacional Autonoma de México. The authors
also thank the Oxford Forestry Institute and A. Sing for providing seed of
Leucaena, J. Pannell for helpful discussions, and L. Urban for comments
on the manuscript. Components of this project were completed while
C.D.B. was on sabbatical at the Department of Plant Sciences, Oxford. This
research was supported by funds from NSF DEB0817033 & EF0542228
(C.D.B.), the Leverhulme Trust (C.E.H.), the Royal Society (C.E.H.), and
the United Kingdom Department for International Development (C.E.H.).
5 Author for correspondence (e-mail:[email protected])
doi:10.3732/ajb.1100259
recognized in land plant evolution, in part because it violates
assumptions associated with bifurcating species trees, but more
importantly because of the evolutionary novelty introduced by
such events (e.g., Rieseberg, 1995; Rieseberg et al., 2003;
Linder and Rieseberg, 2004). As a result, much research focused on recovering the evolutionary history of plant lineages
seeks to distinguish between divergent and reticulate mechanisms and to quantify their relative contributions to the generation of species diversity. At the same time, few studies have
investigated speciation in relation to geography, making it difficult to assess the relative frequency of allopatric vs. sympatric
speciation or the extent to which speciation is associated with
ecological differences (ecological speciation) (Barraclough et al.,
1998; Barraclough and Vogler, 2000).
Here, we investigated patterns of diversification among diploid members of the mimosoid legume genus Leucaena, which
currently comprises 17 diploid species, five tetraploid species,
and a potentially complex series of putative hybrids (Hughes,
1998a) whose evolutionary histories have been influenced by
human translocation of seed, cultivation, and subsequent spontaneous hybridization (Hughes et al., 2002, 2007). All species
of Leucaena are small to medium-sized trees growing predominantly in the seasonally dry tropical forests of Mexico and Central America and extending north into the dry subtropics of
northern Mexico and Texas, and south into the seasonally dry
American Journal of Botany 98(12): 2049–2063, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America
2049
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forests on both sides of the northern Andes as far south as Peru
(Hughes, 1998a). Seeds and pods of a subset of Leucaena species are widely used as a minor food plant in south-central Mexico (Hughes, 1998b; Hughes et al., 2007), and one species, L.
leucocephala has been extensively introduced throughout the tropics as a fast-growing agroforestry and forage tree (Brewbaker,
1987; Hughes, 1998b) and is now a pantropically naturalized,
invasive weed (Hughes and Jones, 1999).
Previous phylogenetic studies of Leucaena, primarily applying data from cpDNA and nrDNA ITS, have failed to fully resolve the relationships among diploid species, conclusively
identify the parentage of several tetraploid species (Hughes et al.,
2002), or investigate the potential occurrence of homoploid
hybridization among diploids. Sequence data from a set of conserved low-copy nuclear genes identified specifically for legume phylogenetics (Choi et al., 2006), as well as a selection of
other low-copy nuclear genes, have proved insufficiently variable within Leucaena to be especially useful (R. Govindarajulu,
unpublished data; Bailey et al., 2004). To overcome these difficulties, we developed a set of anonymous nuclear-encoded
loci identified using a sequence-characterized amplified region
(SCAR) technique that has considerable potential for addressing evolutionary questions in Leucaena (Bailey et al., 2004).
These new SCAR markers and an AFLP-based population
genetic approach are used here to analyze evolutionary relationships among the diploid species of Leucaena and their patterns of diversification with three main objectives. First, diploid
species of Leucaena are the fundamental units from which
polyploids were likely derived, suggesting that a “diploids first”
approach (e.g., Brown et al., 2002; Beck et al., 2010) to resolve
the evolutionary history of diploids is needed to provide foundations for comprehensive downstream assessment of polyploid
origins. The relationships and origins of the polyploid species
are investigated in the accompanying paper (Govindarajulu
et al., 2011 in this issue). Second, it has been unclear to what extent
hybrid origins of several polyploid Leucaena (Hughes et al.,
2002, 2007) might extend to the origin and diversification of
diploids. To examine these questions, we used densely sampled
gene tree and species tree phylogenetic approaches to explore
levels of hybridization and relationships among diploid populations. Third, this framework also served as a molecular test of
the morphologically based species boundaries established by
Hughes (1998a). Resulting diploid species relationships were
then used to guide relevant population genetic comparisons to
further investigate hybridization among diploids and to identify
genetically distinct and isolated population systems corresponding to species.
MATERIALS AND METHODS
DNA extractions—A combination of previously extracted DNA samples
(Bailey et al., 2004; Hughes et al., 2002, 2007) and newly obtained silica-dried
or fresh leaf materials were used for both the DNA sequencing and AFLP studies. DNA recovery applied the chemistry and machinery presented in Alexander et al. (2007), except that 10 mmol/L Tris-HCl in 70% ethanol was used in
place of the 70% ethanol column wash step. DNA quality and quantity were
evaluated by visualizing 3 µL of each sample alongside a 100-bp DNA mass
ladder (NEB-N3231: New England Biolabs, Beverly, Massachusetts, USA) on
1% agarose gels.
Phylogenetic studies using DNA sequence data—Sampling —Two or more
representatives from each of the 17 diploid species and all infraspecific taxa
previously recognized for three of these species of Leucaena were sampled,
making 59 ingroup accessions (Appendix 1). Desmanthus fruticosus and
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Schleinitzia novoguineensis were chosen as outgroups based on the results of
previous studies (e.g., Hughes et al., 2003). Multiple alleles derived from the
same accession are indicated by a numerical suffix (e.g., 1, 2...). For a few accessions, DNA extractions and silica gel dried materials became depleted during the study. These were replaced by DNA from either the same individual tree
or another individual from the same population depending on availability.
PCR, DNA sequencing, and alignment —A total of four cpDNA regions and
seven potentially independent nuclear-encoded loci were sequenced from each
accession. Chloroplast regions sequenced included the two trnK introns flanking the matK gene (primers trnK1L-849R and 1908F-trnK2R, Lavin et al.,
2000), the intron between psbA-trnH (primers psbAF and trnHR; Sang et al.,
1997), and the rpl32-trnL spacer (primers trnL and -rpl32-F, Shaw et al., 2007).
The nuclear-encoded loci included nrDNA ITS and six anonymous SCARbased markers referred to as 23L, 28, A9, A2, PA1213, and A4A5 specifically
developed for Leucaena phylogenetics (Bailey et al., 2004).
PCR reactions included 1× PCR buffer (10 mmol/L Tris-HCl, 50 mmol/L
KCl, 2.5 mmol/L MgCl2), 100 µmol/L of each dNTP, 0.5 µmol/L of each forward and reverse primer, 35 mmol/L betaine, 1.5 U of Taq polymerase and 1 µL
of genomic DNA in a 25-µL reaction. PCR amplifications began with a 3 min
denaturation at 94°C, followed by 35 cycles of 15–30 s denaturation at 94°C,
30–90 s annealing at 57–60°C (see Appendix S1 in Supplemental Data with the
online version of this article) for primers and annealing temperatures), and
60–90 s extension at 72°C; followed by a final extension for 7 min at 72°C. All
of the sequences for chloroplast regions and most of the sequences for SCAR
based loci were generated by direct sequencing of PCR amplified products.
However, accessions that yielded polymorphic reads from the direct sequencing were cloned following previously published methods (Hughes et al., 2002).
As many as 10 colonies were sequenced for each cloned sample to recover
discrete variation consistent with the observed polymorphisms in the directly
sequenced PCR product.
Sequence assembly and phylogenetics —Individual loci were aligned in the
program CLUSTAL_X (Thompson et al., 1997) and manually adjusted by eye
in the program WinClada (Nixon, 2002). For parsimony analyses, indels were
scored as gap characters using the simple gap coding method of Simmons and
Ochoterena (2000) implemented in the program SeqState ver. 1.4 (Müller,
2006). Parsimony analyses were performed with the program NONA (Goloboff,
2000) spawned from WinClada (best tree search, 100 random replications,
holding 10 trees per rep, and applying max* and 1000 strict consensus bootstrap replicates, each comprising 100 mults holding 10 trees each). The best
fitting maximum likelihood (ML) tree (model GTR+Γ) and 500 ML bootstrap
analyses (model GTR+CAT) were performed using the program RAxML
(Stamatakis et al., 2008). Phylogenetic analyses included investigation of individual gene trees and simultaneous analysis of concatenated matrices. First, parsimony
analyses were run on each data partition to assess potential gene tree/species
tree problems and the utility of each locus to resolve relationships within Leucaena. For each gene tree, the positions of multiple accessions from each taxon
and individual sequences from heterozygous individuals were characterized as
monophyletic, “unresolved” (if they were unresolved relative to one another),
or polyphyletic. Attention was particularly paid to “polyphyletic” sequences
from individual accessions because these could indicate potential hybridization
or gene tree/species tree issues. A series of simultaneous analyses were performed including and excluding accessions that were polyphyletic in the individual analyses to evaluate their influence on the topology of the inferred
species tree. However, these accessions did not have any major impacts on support or resolution among diploid species. For the simultaneous analyses presented here, sequences from heterozygous accessions were fused (using IUPAC
ambiguity coding to score polymorphisms) irrespective of whether they were
monophyletic, unresolved, or polyphyletic in the individual analyses to ensure
matching of terminals across data partitions prior to concatenation of matrices.
AFLP studies—Sampling —A total of 424 diploid individuals were subject to
AFLP analysis. Samples for this component included the diploid accessions
used in the phylogenetic study (see above) and DNA extracted from at least 10
individuals from each of 42 populations of diploid Leucaena species (Appendix
1). The latter were derived from greenhouse grown seeds collected from each
of 10 different trees per wild population (Hughes, 1998b).
AFLP analysis—Five positive control samples were included on each 96well plate (L. collinsii subsp. zacapana 57/88/06, L. collinsii subsp. collinsii
51/88/06, L. lempirana 5/91/05, L. multicapitula 81/87/06, and L. salvadorensis
December 2011]
Govindarajulu et al.—Cryptic diversity and allopatry
99/90/02). Restriction ligation reactions (RLs) and preselective amplifications
followed a modified Vos et al. (1995) AFLP approach marketed by Applied
Biosystems (“Plant Mapping Protocol” – P/N 402977 rev. E; Foster City, California, USA). Approximately 50 ng of gDNA was restriction digested using
EcoRI and MseI (New England Biolabs) and ligated with EcoRI and MseI
adaptors using T4 DNA Ligase (New England Biolabs) at 37°C for 12–16 h.
The RLs for each sample consisted of an 11-µL reaction containing 1× T4 Ligase buffer (NEB), 50 mmol/L NaCl, 0.05 mg/mL BSA, 1 pmol/L MseI Adapter
Pair, 10 pmol/L EcoRI Adapter Pair, 1 U MseI, 5 U EcoRI, and 67 U of T4 ligase (NEB). The RLs were subsequently diluted to a final volume of 200 μL
with 0.1× Tris-EDTA (TE). Each sample was subject to preselective amplification with a single selective base on each primer (EcoRI-A and MseI-C) and
three independent selective primer amplifications (5′FAM- EcoRI-AC/MseICTA, 5′FAM-EcoRI-AT/MseI-CTG and 5′JOE -EcoRI-AA/MseI-CTA). Preselective and selective amplifications included 1.5 mmol/L MgCl2, 0.1 mol/L
Tris-HCl pH 8.3, 0.5 mol/L KCl, 0.25 µmol/L of each primer, and ca. 2 U Taq
in a 20-µL reaction containing 4-µL of dilute RL or preselective amplified product, respectively. Cycling conditions followed the ABI Plant Mapping Protocol.
Selective products were capillary electrophoresed on an automated ABI 3100
sequencer (Applied Biosystems) with the Genescan-500 ROX standard
(Applied Biosystems).
AFLP data analysis—The program GeneMapper 4.0 (Applied Biosystems)
was used to identify and score loci between 75 and 500 bp. The total number of
alleles amplified in an accession was compared to the mean and standard deviation of the number of fragments amplified for all individuals for that population
to identify those that failed to amplify well. In practice, those falling below
a standard deviation typically failed to amplify more than few alleles per
reaction.
The data matrix was subjected to distance-based and model-based analyses
to identify distinct genetic clusters among populations. First, principal coordinate analyses (PCO) based on Euclidean distances were generated in the program MVSP ver.3.13m (Kovach Computing Services, Pentraeth, UK). The
PCO analyses display the clustering pattern of genotypes indicating genetic
differentiation among populations and have proved very useful in helping to
identify hybridization at a variety of taxonomic levels when compared to treebased and network-based approaches (Reeves and Richards, 2007). Second, for
subsequent comparisons of specific interest, the number of uniquely supported
genetic clusters was estimated using the model based Bayesian statistical analysis of Pritchard et al. (2000). The scoring of AFLP profiles for the program
structure ver. 2.3.1 treated unobserved alleles as missing data (Evanno et al.,
2005). Structure analyses included 10 000 burn-ins and MCMC replicates for
each run, 10 replicate runs for each K value (K = 1–10). The admixture model
and allele frequencies were treated independently. Finally, the number of
unique genetic clusters was tested by using ΔK calculation as applied by Evanno
et al. (2005).
Geographic distributions and sympatry—To investigate geography and
quantify sympatry in relation to species delimitation and potential reticulate
evolutionary history, we mapped the geographic distributions of (1) the taxa in
each of three major diploid clades, (2) pairs of well-supported recently derived
sister species (sensu Barraclough and Vogler, 2000), and (3) levels of sympatry
observed across the native range of Leucaena irrespective of phylogenetic relationship. Coordinate files for plotting distributions and number of species per
Table 1.
2051
grid cell were generated from a total of 1652 georeferenced herbarium specimen records of diploid Leucaena (examined by Hughes, 1998a) using the program BRAHMS ver. 6.60 (Filer, 2008) and plotted in the program DIVA ver.
7.1.7 (Hijmans, 2010). Sympatry was mapped using a variety of grid cell sizes,
but cell size was found to have little impact on the results, and a 5-km grid cell
size (25 km2) was selected based on estimated pollinator dispersal distances
(see also Hughes et al., 2007).
RESULTS
Phylogenetic analyses of DNA sequences— Extensive PCR,
cloning, and sequencing recovered at least one allele for 636 of
the 649 sequence/accession combinations for the 11 sequenced
regions and 59 ingroup samples. In contrast, presumed primer
site divergence (Bailey et al., 2004) limited the successful PCR
and sequencing in the outgroups Desmanthus fruticosus and
Schleinitzia novoguineensis to just the cpDNA/ITS/PA1213
and cpDNA/ITS loci, respectively. Diploid gene trees lacking
appropriate outgroup sequences were rooted with L. cuspidata
for gene tree comparisons. This rooting derives from L. cuspidata being the only diploid resolved as sister to other Leucaena
in a previous nuclear-based phylogeny of the group (e.g.,
Hughes et al., 2002). Separate analyses of each nuclear-encoded
DNA sequence region and the combined cpDNA matrix were
run to evaluate potential gene tree/species tree conflicts. The
length of each alignment, number of accessions lacking sequence coverage, number of gap characters, percentage parsimony informative characters, and the tree statistics for each data
matrix are presented in Table 1. Assessments of well-supported
nodes confirmed that all data partitions (Appendix S2A–F,
see online Supplemental Data), except A4A5 and nrDNA ITS
(online Appendix S2G, H), were generally congruent with each
other and with results from previous studies (e.g., Hughes et al.,
2002, 2007). However, additional sampling for A4A5 and
nrDNA ITS revealed deep gene tree/species tree problems
manifest by occurrences of highly supported divergent alleles
from single accessions, more than two alleles in many accessions (R. Govindarajulu, unpublished data), and well-supported
incongruence relative to the other loci and one another. These
two loci were excluded from the simultaneous analysis.
Gene trees constructed from separate analyses of chloroplast
and nuclear DNA sequences (Appendix S2) were generally
consistent with results from previous studies using two loci
(Hughes et al., 2002) and, where support was recovered, these
resolved three major diploid clades within Leucaena. From
the 295 sequence/accession combinations that are possible for
the five nuclear-encoded loci and 59 ingroup accessions, we
Summary of data assembled for chloroplast and nuclear gene regions. The concatenated matrix includes all data matrices except A4A5 and
ITS.
Gene region
cpDNA
23L
28
A9
PA1213
A2
A4A5
ITS
Concatenated matrix
Length (bp)
2597
874
742
1235
810
705
778
537
6960
Gap char
104
75
72
156
84
34
77
40
525
No. of PIC
176
160
108
432
121
97
326
328
1100
% PIC
IC
L
CI
RI
6.5
16.8
13.2
28
13.5
12.9
38
55
15.8
1
0
0
3
1
6
NA
NA
NA
178
315
286
1159
326
179
825
895
2355
0.50
0.60
0.44
0.47
0.53
0.69
0.54
0.54
0.54
0.76
0.83
0.75
0.74
0.80
0.90
0.90
0.79
0.81
Notes: PIC, parsimony informative characters; IC, number of accessions without sequence coverage; L, tree length; CI, ensemble consistency index; RI,
ensemble retention index.
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Fig. 1. Best maximum likelihood (ML) tree recovered from simultaneous analysis of five nuclear loci and cpDNA data analyzed with RAxML. Branch
support values represent ML and parsimony derived bootstrap values, respectively.
December 2011]
Govindarajulu et al.—Cryptic diversity and allopatry
2053
Fig. 2. Summary of results for Leucaena lanceolata s.l. (A) PCO scatter plot for all accessions analyzed using AFLPs. (B) Plot of the geographic
distribution of accessions representing divergent groups recovered from phylogenetic analysis (Fig. 1) and PCO (Fig. 2A). (C) Plot of the mean likelihood
estimates calculated for K = 1–10 in structure (Pritchard et al., 2000). (D) ΔK plot calculated according to Evanno et al. (2005).
identified just four cases were two alleles recovered from the
same accession resolved in divergent positions, contradicting
the monophyly of the species (L. collinsii subsp. zacapana
[PA1213; accession 18/84], L. magnifica [PA1213; 19/84], L.
lanceolata var. lanceolata [23L; 134/92], and L. salvadorensis
[A9; 34/88]). Each case was restricted to a single locus for the
respective accession, and the inclusion or exclusion of these
accessions and sequences had no impact on supported nodes in
the combined phylogenetic analysis (R. Govindarajulu, unpublished data).
Both the ML and parsimony-based simultaneous analyses of
the concatenated matrix recovered robustly supported relationships among lineages and provided stronger support and resolution within clades 2 and 3 (Fig. 1) than individual gene trees
(Appendix S2). The modest support for a few nodes within
clade 1 may be attributable to limited character data supporting
these nodes or conflicting signal resulting from the potential
inclusion of hybrid accessions or species. An assessment of unambiguously optimized character distributions along branches
in the parsimony tree suggests that poorly supported nodes are
subtended by short branches relative to other branches in the
combined phylogeny (Appendix S3), further reducing concerns
about the inclusion of hybrid terminals.
With the exception of multiple accessions representing L.
lanceolata, ML and parsimony-based simultaneous analyses
resolved multiple accessions of each diploid or infraspecific
taxon into well-supported monophyletic groups (Fig. 1). In
contrast, the polyphyletic placement of L. lanceolata accessions
from Oaxaca, Veracruz, and western Chiapas (913, 134/92,
43/85, 50/87, 51/87) relative to those from northwestern coastal
Mexico (46/85, 44/85, 90/92) contradicts the current division of
L. lanceolata into two infraspecific varieties proposed by
Hughes (1998a).
AFLP-based assessments— The potential for interspecific
hybridization among diploid species exhibiting few genetic
crossing barriers (Sorensson and Brewbaker, 1994) and problems with the current delimitation of L. lanceolata (see above)
prompted our investigation of species limits using extensive
sampling of individuals and a population genetic approach. The
complete matrix included 1215 loci scored from 363 samples
that amplified well for all three selective primer combinations.
An extensive series of PCO analyses was carried out using the
diploid phylogeny as the framework for relevant focused comparisons. These similarity-based comparisons included clade
to clade analyses (clade 1 to 2, clade 1 to 3, and clade 2 to 3),
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Fig. 3. Summary of results for Leucaena collinsii s.l. (A) PCO scatter plot for all accessions analyzed using AFLPs. (B) Distribution of accessions
representing divergent clades recovered from phylogenetic analysis (Fig. 1) and divergent clusters recovered from PCO (Fig. 3A). (C) Plot of the mean
likelihood estimates calculated for K = 1–10 in structure (Pritchard et al., 2000). (D) ΔK plot calculated according to Evanno et al. (2005).
followed by infraclade comparisons removing the most divergent
taxon(a) sequentially, and ultimately the analysis of accessions
of each species in isolation from other species. These comparisons also recovered distinctive clusters of taxa (online Appendix S4) that were largely consistent with the phylogenetic
hypothesis. In all 27 comparisons only four of 363 accesssions
(L. esculenta 2143 [Appendix S4E], L. lanceolata 1577 [Appendix S4A, D, N, U], L. pueblana 125_92 [Appendix S4B, E,
L], L shannonii 1_91_03 [Appendix S4P]) failed to resolve
with their respective taxon in one or more result, revealing potential hybrid backgrounds or otherwise unique genotypes. This
result along with limited evidence for hybrids in the phylogenetic analyses suggests that we recovered few individuals with
mixed backgrounds indicative of homoploid hybridization
among diploid species.
For L. lanceolata and L. collinsii, more extensive geographic
sampling in the AFLP study revealed levels of genetic diversity
potentially consistent with previously overlooked species-level
diversity. AFLP data from eight populations and 39 accessions
of L. lanceolata were analyzed to investigate the polyphyly of
L. lanceoata observed in the combined phylogenetic analysis
(Fig. 1). PCO analyses resolved L. lanceolata accessions into
two distinct groups (44/85, 46/85) and (134/92, 43/85, 51/87,
2166, 2171, 1577) along axis 1 (Fig. 2A) and are further supported by results recovered from structure (ΔK = 2; Fig. 2C,
D). These groups correspond to the two divergent clades recov-
ered in the phylogenetic analysis (Fig. 1), and the clusters differ
by six fixed alleles in the AFLP data set. Leucaena lanceolata
var. lancolata accession 1577 showed evidence of possible hybridization between these two lineages, as evidence by an admixed background in results from structure and its
intermediate position in PCO plots (Fig. 2). However, 1577 behaved erratically in each comparison that it was included (Fig.
2 and Appendices S4A, D, N, U), even resolving between clades
1 and 3 in broad comparisons. The cause of these anomalous
placements is unclear.
Separate PCO analyses of multiple populations representing L. collinsii subsp. collinsi and L. collinsii subsp. zacapana, which formed strongly supported monophyletic sister
groups in the phylogeny (Fig. 1), resolved these taxa into
divergent clusters (Fig. 3A). These were also recovered from
structure (ΔK = 2, Fig. 3C, D), and they differed by nine
fixed alleles. None of these results identified potential admixed individuals.
Genetic diversity consistent with potential overlooked species was also recovered in PCO analyses of available material
of L. lempirana (Appendix S4Y), L. macrophylla (Appendix
S4W), L. salvadorensis (Appendix S4Z), and L. trichodes (Appendix S4X). The single populations of L. trichodes sampled
from the east and west sides of the Andes in Venezuela and
Ecuador, respectively, differed by 36 fixed allelic differences;
samples from one population each for the subspecies for L.
December 2011]
Fig. 4.
Govindarajulu et al.—Cryptic diversity and allopatry
2055
Geographic distribution of 1652 herbarium records representing diploid accessions of Leucaena from clades 1–3.
macrophylla differed by 12 fixed allelic differences; two populations of L. lempirana from closely adjacent valleys in northern Honduras differed by seven fixed allelic differences; and
populations of L. salvadorensis from northern Nicaragua differed by three fixed allelic differences from those in southern
Honduras. However, all these comparisons are based on sparse
sampling across the geographic range in both the phylogenetic
and population genetic approaches and may thus be biased.
Geographic distributions— Mapping of 1652 georeferenced
wild diploid herbarium specimen records using the three different approaches shows a clear pattern of allopatry among diploid
species. First, the geographic distributions of the three major
clades, with minor exceptions, are almost entirely allopatric
(Fig. 4) with taxa from clade 3 restricted to northeast Mexico
and the southern United States, areas east and north of central
volcanic axis across Mexico, taxa from clade 2 restricted to inland areas of south-central Mexico in the central Mexican highlands and valleys south of the volcanic axis, while taxa belonging
to clade 1 occur along the western coast of Mexico into southcentral Mexico, Central America, and northern South America
(Fig. 4). The geographically widespread clade 1 species L. macrophylla generates a slight (Appendix S5), and in some areas
superficial (as a result of the scale), overlap in distribution between clade 1 and clade 2 taxa (Fig. 4).
An assessment of sympatry irrespective of phylogenetic relationship identified very few regions with potential overlap among
noncultivated wild diploid accessions, with just 30 of the 1104
grids (5-km grid cells) containing populations of two diploid species (Fig. 5; Appendix S5) and none containing more than two.
Finally, geographic distributions of each well-supported pair
of sister species are also generally allopatric. For example,
Fig. 5. Geographic representation of sympatry among all 1652 diploid accessions of Leucaena. Each symbol on the map represents one of 1104 grid
cells that had one or more occurrences of Leucaena; either a single species or two species occurred in that 25-km2 area.
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Fig. 6. Geographic distributions of well-supported sister species pairs recovered from phylogenetic analysis (Fig. 1). (A) Leucaena greggii and L.
retusa. (B) L. multicapitula and L. salvadorensis. (C) L. matudae and L. pueblana. (D) L. lanceolata s.s. and L. macrophylla.
L. greggii/L. retusa (Fig. 6A), L. multicapitula/L. salvadorensis
(Fig. 6B), L. collinsii subsp. collinsii/L. collinsii subsp. zacapana (Fig. 3B), and L. matudate/L. pueblana (Fig. 6C) occupy
strictly allopatric extant distributions. Only the pair of sister
species L. lanceolata (s.s.) (see below) and L. macrophylla are
found in partial sympatry (Fig. 6D).
DISCUSSION
Previous investigations of phylogenetic relationships among
species of Leucaena and its close relatives (e.g., Luckow, 1997;
Hughes et al., 2002, 2003) have run into common and recurrent
problems associated with limited available variation at some
universally applied loci (e.g., Shaw et al., 2007) and limited
concerted evolution with nrDNA ITS (e.g., Álvarez and
Wendel, 2003). The application of the SCAR-based approach
of Bailey et al. (2004) for locus development, specifically
implemented for studies involving closely related species, has
proven useful in resolving highly supported divergent diploid
species relationships in Leucaena. This finding is also consistent with the application of (Thorogood et al., 2009) and at least
one extension of the method (González, 2010) in other plant
groups. Thus this approach, along with conserved orthologous
markers (e.g., Fulton et al., 2002; Choi et al., 2006; Lohithaswa
et al., 2007), has helped bridge the gap between sole reliance on
standard phylogenetic markers (e.g., nrDNA ITS and certain
cpDNA markers) and the coming accessibility of massive
marker sets derived from second generation sequencing approaches in nonmodel systems (e.g., Gompert et al., 2010;
M. M. Koopman [Eastern Michigan University] et al., unpublished manuscript).
Species diversity—Inclusion of multiple accessions of all species in the phylogenetic analyses and population genetic assessments, along with detailed distribution maps for all taxa, provide
an excellent basis for the re-evaluation of boundaries among
diploid species of Leucaena. For 16 of the 17 species recognized
by Hughes (1998a), we found high support for monophyly of multiple accessions in the phylogenetic analyses that is congruent with
results from population genetic assessments, morphology, and
geographic isolation among wild populations, supporting the
species circumscriptions presented in the monographic treatment of Hughes (1998a). These results are consistent with a variety of species concepts, including the phylogenetic species
concept (sensu Nixon and Wheeler, 1990; Davis and Nixon, 1992),
monophyletic species concept (e.g., Donoghue, 1985), and biological species concept (Dobzhansky, 1935; Mayr, 1942).
These data and analyses also provide strong evidence for previously overlooked cryptic species within L. collinsii and L.
lanceolata. The two divergent and well-supported monophyletic clades of accessions representing L. lanceolata (Fig. 1),
which are congruent with population genetic differences (Fig. 2),
contradict current and historical species delimitations. However, these lineages occupy distinct and disjunct geographic
distributions (Fig. 2), providing further evidence for two distinct species. Similarly, accesssions of L. collinsii subsp. zacapana
are geographically isolated from L. collinsii subsp. collinsii
(Fig. 3), and the two subspecies formed well-supported monophyletic groups (Fig. 1) for which fixed allelic differences were
detected in the population genetic analyses (Fig. 3). These findings along with morphological and chromosome differences
support recognition of these as distinct species (see taxonomic
treatment later), increasing the number of recognized diploid
species of Leucaena from 17 to 19.
December 2011]
Govindarajulu et al.—Cryptic diversity and allopatry
In addition to these clear-cut examples of previously underestimated species diversity, population genetic assessments reveal population structures and fixed allelic differences indicative
of strongly differentiated and geographically structured variation among populations of several other species of Leucaena,
including L. lempirana, L. macrophylla, L. salvadorensis, and
L. trichodes. This variation is especially notable within L. trichodes
where populations from opposite sides of the Andes in Venezuela and Ecuador differ by 36 fixed allelic differences, reflecting
the likely lack of gene exchange and degree of isolation across
the Andes (e.g., Dick et al., 2003). However, much denser sampling in both the phylogenetic and population genetic analyses
would be needed to confidently distinguish whether these patterns are the result of sparse sampling or cryptic evolutionary
divergence among populations that could merit recognition of
additional species.
The discovery of overlooked species diversity prompted by
densely sampled (complete or near-complete taxon sampling and
multiple accessions of species) molecular phylogenetic analyses
that reveal robustly supported reciprocally monophyletic clades
that coincide with other evidence from geography, ecology, and
morphology is increasingly common. Taking examples of legumes from neotropical seasonally dry tropical forests, recent
novelties delimited in similar ways include Caesalpinia oyame
(Sotuyo et al., 2007; Sotuyo and Lewis, 2007), Mimosa jaenensis
(Särkinen et al., 2011), Coursetia greenmanii (de Stefano et al.,
2010), Coursetia caatingicola (de Quieroz and Lavin, 2011), and
Poissonia eriantha (Pennington et al., 2011). This steady addition of new species across different genera suggests that species
diversity of neotropical seasonally dry forests may have been significantly underestimated.
Hybridization and speciation among diploid Leucaena— At
the diploid level, the evolutionary significance of homoploid
hybridization and introgression in species diversification has
remained controversial (e.g., Anderson and Stebbins, 1954).
The role of hybridization in the formation of polyploid species
and lineages is widely recognized (e.g., Doyle et al., 1990;
Wendel et al., 1995); however, there is growing evidence that
homoploid hybridization and introgression have also been important in many plant and animal groups (e.g., Baack et al.,
2005; Kane et al., 2009). In Leucaena, the important outcomes
of hybridization in terms of multiple polyploid taxa are clearly
established (Hughes et al., 2002, 2007; Govindarajulu et al.,
2011 in this issue), and a variety of studies have demonstrated
the potential for hybridization among diploids in experiments
that show high artificial crossability between species (e.g., Sorensson and Brewbaker, 1994) and as a result of human translocation and cultivation (Hughes et al., 2007). Nonetheless, our
results derived from individual gene tree hypotheses, combined
species tree hypotheses, and an AFLP-based population genetic
approach revealed little evidence for hybridization or introgression between wild-collected diploid individuals or populations,
suggesting that reticulation has been of little importance in the
historical diversification of diploid Leucaena. Furthermore, the
predominantly allopatric geographic distributions (Figs. 4–6)
appear to confirm that there are few opportunities for diploid hybrids to arise in wild populations. Thus, the available evidence
suggests that diploid Leucaena are predominantly derived from
divergent, rather than reticulate, mechanisms of speciation.
Such divergent modes of speciation are partitioned into allopatric or sympatric-parapatric mechanisms. Allopatric speciation, long considered the most common mechanism of speciation
2057
in plants and animals (e.g., Mayr, 1942), entails geographic isolation between populations and subsequent divergence into distinct
lineages. In contrast, sympatric and parapatric speciation is inferred for species with overlapping or proximate distributions
during speciation, which require the development of reproductive isolating mechanisms as part of the speciation process (e.g.,
Kondrashov, 1986). Although few studies have tested the relationship between geography and speciation (e.g., Barraclough
and Vogler, 2000; Savolainen et al., 2006; Papadopulosa et al.,
2011), where such data are available for non-island systems, cladogenesis has been found to be predominantly associated with
allopatry, with sympatry interpreted as a consequence of postspeciation range movements (Perret et al., 2007). In contrast, recent
studies that focused on island and island-like systems have begun
to question predominant views on the relative importance of allopatric and sympatric mechanisms of speciation (Barluenga et al.,
2006; Savolainen et al., 2006; Papadopulosa et al., 2011).
Contemporary geographic distributions of diploid species of
Leucaena show a high degree of allopatry consistent with allopatric divergent speciation as the predominant mechanism underlying diploid species diversification in Leucaena (e.g., Grant,
1971; Barraclough and Vogler, 2000). Allopatry is evident at
three levels: (1) the early divergence of major clades whose
taxa remain largely allopatric (Fig. 4), (2) overall patterns of
wild populations of diploid species irrespective of their phylogenetic relationships (Fig. 5), and (3) four of the five recently
derived well-supported pairs of sister species (Figs. 3, 6).
Furthermore, artificial crossing experiments in Leucaena
have shown a low degree of reproductive isolation between
most diploid taxa. The results of 65 diploid-diploid crosses,
representing 12 of the 19 species analyzed here, show a high
degree of crossability (77%) (Sorensson and Brewbaker, 1994).
Although crossability remains to be tested among the remaining diploids, currently available data show that crossability is
retained among distantly divergent lineages across the whole
diploid phylogeny. The overwhelming absence of diploid-diploid hybrids in the context of genus that retains crossability is in
line with geographical isolation and allopatry underlying diploid species diversification in the genus.
Biogeography— Species of Leucaena are concentrated in the
seasonally dry tropical forest (SDTF) biome, a vegetation type
that occupies a wide but highly disjunct distribution across the
neotropics (Pennington et al., 2000, 2009) and is characterized
by erratic moisture availability, long periods of seasonal
drought, a general absence of grasses and natural fire disturbance, high levels of endemism (β diversity), and an abundance
of succulent plants including Cactaceae that has prompted its
designation as the “succulent biome” (Schrire et al., 2005). A
predeliction for this type of vegetation suggests that Leucaena
shows a pattern of phylogenetic niche conservatism (sensu
Donoghue, 2008) to SDTFs, with only minor incursions of a
few lineages into mid-elevation seasonal pine–oak forests
(L. trichandra and L. macrophylla), more-mesic less-seasonal
lowland forests (L. multicapitula), and subtropical dry matorral
(L. greggii and L. retusa). This distribution pattern suggests
that diversification of diploid Leucaena was determined more
by geographic than ecological isolation, in line with the predominance of allopatry among species.
In common with phylogenies of other woody SDTF clades,
the three major clades within Leucaena occupy distinct and
largely disjunct geographical areas in northeast Mexico and
Texas, inland south-central Mexico, and Pacific coastal Mexico,
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American Journal of Botany
Central America, and northern South America (Fig. 4). This
pattern of strong geographical structuring in the diploid Leucaena
phylogeny, and across phylogenies for other dry forest groups
(Lavin, 2006), has been attributed to limited dispersal and immigration across the fragmented SDTF biome (Lavin, 2006;
Pennington et al., 2009), a pattern potentially accentuated by
the resilient ecology of dry forests and phylogenetic niche conservatism (Pennington et al., 2010).
Coalescence of sequences of nuclear loci and the resultant reciprocal monophyly of multiple accessions of Leucaena species is
also consistent with patterns observed for other seasonally dry
tropical forest lineages and indicative of long persistence of endemic populations and allopatric speciation in geographically
isolated and evolutionarily persistent dry forest patches (Barraclough, 2010; de Stefano et al., 2010; Pennington et al., 2010).
Conclusions— We find little evidence for contemporary or
historical hybridization among wild-collected diploids and, as a
result of limited reticulation and the utility of the markers used,
recover a well-resolved phylogenetic species-level hypothesis.
Population genetic structure and phylogenetic resolution identify two additional morphologically cryptic species that are
supported by a variety of data. Last, the pattern of diversification across neotropical seasonally dry forests is consistent with
a general mechanism of divergent allopatric speciation in the
formation of most diploid species of Leucaena.
Diploid Leucaena represent the majority of species in a
genus known to be complicated by human translocation, polyploidy, and hybridization (e.g., Harris et al., 1994; Hughes and
Harris, 1998; Hughes et al., 2002, 2007). Through the application of newly available data, dense sampling strategies, and
complementary phylogenetic and population genetic approaches, we have clarified the evolutionary diversification of
diploids. There is little evidence to suggest that reticulate evolutionary processes have played a significant role in the diversification of diploid Leucaena.
The recognition of L. cruziana and L. zacapana as cryptic
species is in keeping with a renaissance of species discovery
being driven in part by the development of new tools for DNA
barcoding and concerns related to loss of biodiversity (e.g.,
Savolainen et al., 2005; Smith et al., 2008). Furthermore, the
recovery of cryptic species-level diversity in Leucaena is consistent with densely sampled phylogenies revealing geographically structured genetic variation with patterns of coalescence
among conspecific accessions for a growing number of neotropical seasonally dry forest plant groups (Sotuyo and Lewis,
2007; Pennington et al., 2009, 2010, 2011; de Stefano et al.,
2010; de Quieroz and Lavin, 2011; Särkinen et al., 2011).
Finally, a general pattern of allopatric divergence among
diploid Leucaena species is in line with historical opinion
suggesting that this mechanism is the “null” model of speciation in most sexual lineages (e.g., Coyne and Orr, 2004).
However, this stands in contrast to a growing body of evidence that gene flow can be common during and after speciation in many groups (e.g., Chapman and Burke, 2007;
Papadopulosa et al., 2011) as well as abundant evidence for
allopolyploid origins of several Leucaena species (Hughes
et al., 2002, 2007). Reconciling this pattern of strictly allopatric
diploid speciation with known allopolyploid speciation in
Leucaena reinforces the idea that human translocation and
cultivation have been critical in creating artificial sympatry
and opportunities for hybridization (Hughes et al., 2007;
Govindarajulu et al., 2011 in this issue).
TAXONOMIC TREATMENT
Leucaena zacapana— The results presented here strongly
support raising L. collinsii subsp. zacapana to species rank,
distinct from L. collinsii. Multiple accessions of each taxon
form well-supported monophyletic sister clades (Fig. 1), and
these groups were also recovered in all the population genetic
analyses (e.g., Fig. 3A). These two population systems are further distinguished by nine fixed AFLP allelic differences and
by cytological studies that suggest L. collinsii subsp. colllinsii
is 2n = 52, while L. collinsii subsp. zacapana is 2n = 56 (Cardoso
et al., 2000; Schifino-Wittmann et al., 2000), and by genome
size measurements (Govindarajulu et al., 2011 in this issue). In
addition, a suite of quantitative morphological differences
(Hughes, 1998a) and geographically disjunct and isolated distributions of these two lineages (Fig. 3B), further support recognition as two distinct species.
As recognized here Leucaena collinsii is restricted to the
central depression of Chiapas in Mexico and adjacent fringes of
the Departamento of Huehuetenango in Guatemala, between
400 and 900 m a.s.l., while L. zacapana is a narrowly restricted
endemic in the Motagua Valley system in Guatemala between
100 and 800 m above sea level (fig. 44 in Hughes, 1998a). The
intervening mountains of central and northwestern Guatemala
rising to between 2000 and 3000 m a.s.l., effectively isolate
these two species.
The seasonally dry tropical forests of the Motagua Valley
system in southeastern Guatemala are known to harbor a number of endemic dry forest plant species (e.g., in legumes Calliandra carcera Standl. & Steyermark, Mimosa canahuensis
Standl. & Steyermark, Aeschynomene eriocarpa Standl. &
Steyermark). However, compared to some other neotropical
seasonally dry valleys such as the central depression of Chiapas
and the Tehuacán Valley in south-central Mexico, or the Marañón
Valley in northern Peru, current estimates of endemism for Motagua are modest. Thus, it seems at first sight somewhat surprising
that there are two endemic species of Leucaena, L. magnifica, a
narrowly restricted endemic only known from the Guatemalan
Department of Chiquimula (Hughes, 1998a), and L. zacapana
from this one valley system. These findings suggest that levels
of endemism in the Motagua valley may be underestimated.
Leucaena zacapana (C. E. Hughes) R. Govindarajulu &
C. E. Hughes comb. et stat. nov. Leucaena collinsii subsp.
zacapana C. E. Hughes, Kew Bull. 46(3): 553, 1991. Type:
Guatemala. Zacapa: Estanzuela in dry thorn forest, 1 Mar 1988,
Hughes 1102 (holotype: FHO!; isotypes: K! MEXU!).
As circumscribed here, L. zacapana corresponds directly to
L. collinsii subsp. zacapana presented by Hughes (1998a). Distinguishing features, illustrations, and specimen citations lists
previously presented under L. collinsii by Hughes (122–128
and fig. 43 in Hughes, 1998a) can be directly applied and are
not further elaborated on here.
Leucaena lanceolata s.l.—None of the previous circumscriptions of the variable and widely distributed L. lanceolata has
proved satisfactory. These treatments range from the recognition
of a single taxon (McVaugh, 1987) to division into either nine
separate species (Britton and Rose, 1928) or two infraspecific
taxa (Zárate, 1994; Hughes, 1998a). While several distinct morphological variants are apparent across the range of L. lancoleata
s.l., these are often geographically localized and based on relatively minor quantitative differences in leaves and pods, while
overall patterns in these traits show no clear-cut discontinuities
December 2011]
Govindarajulu et al.—Cryptic diversity and allopatry
that are congruent with geography (figs. 58, 59 in Hughes, 1998a).
Notably, populations from the Pacific coast of eastern Michoacán
and inland populations from Oaxaca show overlapping patterns
of pod size and indumentum spanning the boundaries between
the two infraspecific taxa, L. lanceolata var. lanceolata and L.
lanceolata var. sousae recognized by Zárate (1994) and Hughes
(1998a). We identify two robustly supported lineages that coincide with the geographical disjunction between populations from
western and northwestern Mexico (western Guerrero and Michoacán to Sonora and Baja California) and those from Pacific
coastal Oaxaca, southwestern Chiapas, spanning the Isthmus of
Tehuantepec to Veracruz (Fig. 2B). Geographically structured genetic variation of this sort is a common feature of seasonally dry
tropical forest plants (e.g., Lavin, 2006; Pennington et al., 2009)
and provides a robust basis for delimitation of two species, here
recognized as L. lanceolata (the typical northwestern lineage)
and L. cruziana Britton & Rose for the Oaxaca, Veracruz, and
western Chiapas lineage.
Recognition of L. cruziana as a species distinct from L. lanceolata is congruent with results from analysis of plastid and
nuclear DNA, AFLP data, and geography, and strongly supported by fixed differences indicative of isolation and consistent
with the phylogenetic species concept (see above). Re-examination of the somewhat complex and overlapping patterns of
variation in quantitative leaf and pod traits in the light of these
new results, suggests that this new division is satisfactory in
comparison to previous classifications. The narrower circumscription of L. lanceolata proposed here to include only populations from western Guerrero to Sonora including Baja California
(Fig. 2B), creates a morphologically more coherent species uncomplicated by the variability in pod traits and especially pod
vestiture found in coastal Michoacán and parts of Oaxaca,
which was highlighted as problematic by Hughes (1998a). Leucaena lanceolata, as circumscribed here, generally has leaves
with 3–5 pairs of pinnae, 4–6 pairs of leaflets per pinna and
leaflets <20 mm wide and pods <18 cm long and < 22 mm wide,
while L. cruziana has leaves with 2–3(−4) pairs of pinnae, 3–
4(−5) pairs of leaflets per pinna, leaflets 20–35 mm wide, and
pods (16–)20–37 cm long and (16–)20–32 mm wide, although
there are no clear-cut discontinuities in any of these traits.
The revised synonymy for L. lanceolata listed below is less
extensive than suggested by Hughes (1998a), but still includes
five species described by Britton and Rose (1928), who tended
to pigeon-hole the minor variants they observed among the limited material available to them, as distinct species.
Under this new division, pod indumentum, which was previously used as one character to distinguish typical L. lanceolata
var. lanceolata from L. lanceolata var. sousae, but for which
there were several notable and problematic exceptions (Hughes,
1998a), is confirmed to be an unreliable and labile character for
species delimitation. Pods vary from densely velutinous to glabrous (when pods are often lustrous or glossy) within and
among populations of both L. cruziana and L. lanceolata, just
as it does within several other species of Leucaena (e.g., L. diversifolia, L. lempirana, L. trichandra) (Hughes, 1998a).
Leucaena lanceolata S. Watson, Proc. Amer. Acad. Arts 21:
427. 1886. Type: MEXICO. Chihuahua: Batopilas, Hacienda
San Miguel, SW Chihuahua, 27°53′N, 108°26′W, Sep 1885,
Palmer 6 (holotype: GH!; isotype: NY!UC!US!).
Leucaena microcarpa Rose, Contr. U. S. Natl. Herb. 5: 141.
1897. Type: MEXICO. Baja California Sur: nr Miraflores, 23°21′N, 109°47′W, 13 Oct 1890, Brandegee 186
(holotype: US!; isotype: UC!).
2059
Leucaena brandegeei Britton & Rose, N. Amer. Fl. 23: 128.
1928. Type: MEXICO. Baja California Sur: nr La Mesa,
Cape region, 31 Oct 1902, T.S. Brandegee s.n. (holotype:
NY!; isotypes: US!UC!).
Leucaena palmeri Britton & Rose, N. Amer. Fl. 23: 123.
1928. Type: MEXICO. Sonora: nr Alamos, 26°59′N,
108°57′W, 20 Sep 1890, Palmer 718 (holotype: NY!;
isotype: US!).
Leucaena pubsecens Britton & Rose, N. Amer. Fl. 23: 122.
1928. Type: MEXICO. Sinaloa: nr Mazatlán, 23°14′N,
106°24′W, 1925, J.G. Ortega 5988 (holotype: NY!; isotypes: GH!US!).
Leucaena sinaloensis Britton & Rose, N. Amer. Fl. 23: 124.
1928. Type: MEXICO. Sinaloa: vicinity of Palmar,
22°13′N, 105°36′W, 15 Apr 1910, Rose et al., 14650 (holotype: NY!; isotype: US!).
Leucaena sonorensis Britton & Rose, N. Amer. Fl. 23: 122.
1928. Type: MEXICO. Sonora: Sierra de Alamos, nr
Alamos, 26°58′N, 108°57′W, 14 Mar 1910, Rose et al.,
12821 (holotype: NY!; isotype: US!).
Leucaena nitens M. E. Jones, Contr. West Bot. 15: 136. 1929.
Type: MEXICO. Sinaloa: nr Mazatlán, 23°14′N,
106°24′W, 20 Nov 1926, Jones 22465 (holotype: POM!;
isotypes: MO!US!).
For additional material examined, see online Appendix S6.
Leucaena cruziana Britton & Rose, N. Amer. Fl. 23: 123.
1928. Type: MEXICO. Veracruz: Barranca de Panoaya,
19°18′N, 96°25′W, Dec 1919, Purpus 8387 (holotype: NY!;
isotypes: GH!UC!US!).
Leucaena rekoi Britton & Rose, N. Amer. Fl. 23: 122. 1928.
Type: MEXICO. Oaxaca, nr Pochutla, close to the Pacific coast, 15°44′N, 96°28′W, 28 Sep 1917, Reko 3632
(lectotype, flowering shoot and leaves only: US!).
Leucaena purpusii Britton & Rose, N. Amer. Fl. 23: 123.
1928. Type: MEXICO. Veracruz: Rim of barranca at Remudadero, 19°15′N, 96°34′W, Jan 1926, Purpus 10607
(holotype: NY!; isotype: US!).
Leucaena lanceolata var. sousae (S. Zárate) C. E. Hughes,
Contr. Univ. Michigan Herb. 21: 288. 1997. Leucaena
lanceolata subsp. sousae S. Zárate, Anales Inst. Biol.
Univ. Auton. México, BOT. 65: 117. 1994. Type: MEXICO. Oaxaca: 17 km WNW of Puerto Escondido, Distr.
Juquila, 15°57′N, 97°13′W, 21 Oct 1976, Sousa 6390
(holotype: MEXU!; isotype: UC!).
Three of the nine species previously recognized by Britton
and Rose (1928) and subsequently treated as conspecific with
L. lanceolata by Zárate (1994) and Hughes (1998a)—L. rekoi,
L. cruziana and L. purpusii—as well as the subsequently described L. lanceolata subsp. sousae (Zárate, 1994; Hughes,
1998a), have type localities (from Pochutla, Oaxaca; Barranca
de Panoaya, Veracruz; Remudadero, Veracruz; and Puerto Escondido, Oaxaca, respectively) that fall within the distribution
of the southeastern lineage. The three earlier names by Britton
and Rose (1928) were published simultaneously in their North
American Flora, but doubt has been cast over the identity of L.
rekoi (Zárate, 1994), because the type collection is a mixed
gathering of leaves and flowers from Leucaena, which Zárate
(1994) suggests are doubtfully distinguishable from L. macrophylla, and fruits of Caesalpinia (Coulteria) velutina (Britton
& Rose) Standl. Of the two remaining names, which are both
based on types from geographically closely adjacent localities
in Veracruz, we are choosing to use the name L. cruziana,
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American Journal of Botany
which appears before L. purpusii, albeit on the same page in
Britton and Rose (1928), to recognize the southeastern lineage
as a distinct species.
Denser sampling of accessions, and especially from the
populations in Veracruz where only a single individual is included in this study) will be needed to assess the full extent
of variation across this group and the possibility of further
subdivision.
For additional material examined, see Appendix S6.
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Appendix 1. Plant material used for phylogenetic study and AFLP analyses. Seed lot number is provided if DNA was extracted from a seedling raised from a seedlot.
Herbarium vouchers are all at FHO, with duplicates variously deposited at CAS, EAP, K, MEXU, NY, US, and MO. For complete locality and GenBank
information, see Appendix S7 (in Supplemental Data with the online version of this article).
(A) Phylogenetic study
Taxon, Voucher, Seed lot (where applicable, e.g., 51/81) -Locality.
L. collinsii Britton & Rose, Hughes CE 1187, 51/88 -Huehuetenango,
Guatemala. Hughes CE 527, 52/88 -Chiapas, Mexico. L. cruziana, Hughes
CE 1672, 134/92 -Matias Romero, Oaxaca, Mexico. Hughes CE 2180, -El
Limon, Palmasola, Veracruz, Mexico. Hughes CE 559, 43/85 -Oaxaca,
Mexico. Hughes CE 913, -Veracruz, Mexico. Hughes CE 835, 51/87
-Oaxaca, Mexico. Hughes CE 872, 50/87 -Oaxaca, Mexico. L. cuspidata
Standley, Hughes CE 1580, -Tolantongo Cardonal, Hidalgo, Mexico.
Hughes CE 1583, 89/92 -Mina San Miguel, Hidalgo, Mexico. Hughes CE
1586, 88/92 -Jacala, Hidalgo, Mexico. L. esculenta (Sessé & Mociño ex
DC.) Bentham, Hughes CE 2114, -Miahuatlán, Oaxaca, Mexico. Hughes
CE 2143 -Huajuapan de León, Oaxaca, Mexico. Bailey & Ochoterena 216,
-Mexico, Mexico. Hughes CE 894, 47/87 -Guerrero, Mexico. Hughes CE
903, 48/87/01 -Michoacan, Mexico. Desmanthus fruticosus Rose, Hughes
CE 1532, 109/92 -La Paz,Baja california, Mexico. L. greggii S. Watson,
Hughes CE 1057, 82/87 -Nuevo León, Mexico. Hughes CE 695, 19/86
-Nuevo León, Mexico. Hughes CE 695, 20/86/02 -Nuevo León, Mexico.
Hughes CE 695, 21/86/07 -Nuevo León, Mexico. L. lanceolata S. Watson,
Hughes CE 1577, 90/92 -Alamos, Sonora, Mexico. Hughes CE 631, 46/85
-Michoacán, Mexico. L. lempirana C.E. Hughes, Hughes CE 1411,
6/91/03 -Negrito, Yoro, Honduras. Hughes CE 1447, 5/91 -Aguan Valley,
Yoro, Honduras. L. macrophylla subsp. istmensis C.E. Hughes, Hughes
CE 580, 47/85 -Oaxaca, Mexico. L. macrophylla subsp. macrophylla
Bentham, Hughes CE 1179, 55/88 -Guerrero, Mexico. Hughes CE 2076,
-Coxcatlan, Puebla, Mexico. L. magnifica (C.E Hughes) C.E. Hughes,
Hughes CE 1089, 58/88 -Chiquimula, Guatemala. Hughes CE 412, 19/84
-Chiquimula, Guatemala. L. matudae (S. Zárate) C.E. Hughes, Hughes CE
2153, -San Miguel Tecuixiapan, Guerrero, Mexico. Hughes CE 879, 49/87
-Guerrero, Mexico. L. multicapitula Schery, Hughes CE 1024, 86/87
-Penas Blancas, Guanacaste, Costa Rica. Hughes CE 1025, 81/87 -Los
Santos, Panama. Schleinitzia novo-guineensis (Warb.) Verdc., Chaplin,
57/84 -Munda, Soloman Islands. L. pueblana Britton & Rose, Hughes
CE 1648, 125/92 -Lower Tehuacan Valley, Oaxaca, Mexico. Hughes CE
2089, -Cuicatlan, Oaxaca, Mexico. Hughes CE 2140, -Santo Domingo
Tonala, Oaxaca, Mexico. L. pulverulenta (Schlechtendal) Bentham,
Hughes CE 1051, 83/87/02 -Tamaulipas, Mexico. Hughes CE 1058,
84/87 -Texas, USA. Hughes CE 1593, -Xilitla, San Luis Potosí, Mexico.
Hughes CE 1611, -Huejutla de Reyes, Hidalgo, Mexico. Hughes CE 1866,
-Misantla, Veracruz, Mexico. L. retusa Bentham in Gray, Rajanikanth
& Bailey 2009, 23/09/02 -Eddy Co, New Mexico, USA. Bendeck s.n.,
23/86 -Coahuila, Mexico. L. salvadorensis Standley ex Britton & Rose,
Hughes CE 1407, 7/91 -San Juan de Limay, Esteli, Nicaragua. Hughes
CE 742, 17/86 -Choluteca, Honduras. Hughes CE 746, 34/88 -Choluteca,
Honduras. L. shannonii Donnell Smith, Hughes CE 1417, 1/91 -Santa
Caterina Mita, Jutiapa, Guatemala. Hughes CE 1676, 135/92 -Cintalapa
de Figueroa, Chiapas, Mexico. Hughes CE 1714, 141/92 -Santa Rita,
Yoro, Honduras. Hughes CE 507, 53/87 -Champoton, Campeche, Mexico.
L. trichandra (Zuccarini) Urban, Hughes CE 1106, 53/88 -Guatemala,
Guatemala. Hughes CE 1130, 54/88 -Huehuetenango, Guatemala.
Hughes CE 1421, 4_91 -Erandique, Lempira, Honduras. Hughes CE
1654, 128/92 -Tierra Colorada, Oaxaca, Mexico. Hughes CE 1682,
137/92 -La Trinitaria, Chiapas, Mexico. Hughes CE 1701, 140/92 -San
Marcos, San Marcos, Guatemala. Hughes CE 2121, -Matatlan, Oaxaca,
Mexico. L. trichodes (Jacquin) Bentham, Hughes CE 775, 2/86/07
-Trujillo, Venezuela. Hughes CE 997, 61/88 -Manabi, Ecuador. L.
zacapana (C.E. Hughes) R. Govindarajulu & C.E. Hughes, Hughes CE
1096, 57/88 -Chiquimula, Guatemala. Hughes CE 1120, 56/88 -Zacapa,
Guatemala. Hughes CE 299, 18/84 -Progreso, Guatemala.
(B) AFLP analyses
Taxon, Voucher, Seed lot (where applicable, e.g., 51/81) -Locality, -Number of
individuals analyzed per population.
L. collinsii Britton & Rose, Hughes CE 527, 45/85 -Narcisco Mendoza,
Chiapas, Mexico -8, Hughes CE 1187, 51/88 -Chacaj, Huehuetenango,
Guatemala -13. L. cruziana Britton & Rose, Hughes CE 1672, 134/92
-Matias Romero, Oaxaca, Mexico -1, Hughes CE 559, 43/85 -San jon,
Oaxaca, Mexico -10, Hughes CE 2166, -Cerro Gordo, Veracruz, Mexico
-1, Hughes CE 2171, -La Mancha, Palmasola, Veracruz, Mexico -1,
Hughes CE 835, 51/87 -Puerto Angel, Oaxaca, Mexico -11. L. cuspidata
Standley, Hughes CE 1851, 83/94 -Camarones, Hidalgo, Mexico -9,
Hughes CE 1586, 88/92 -Jacala, Hidalgo, Mexico -1, Hughes CE 1583,
89/92 -Mina San Miguel, Hidalgo, Mexico -1. L. esculenta (Sessé &
Mociño ex DC.) Bentham, Hughes CE 2114, -Miahuatlán, Oaxaca,
Mexico -1, Hughes CE 2143, -Huajuapan de León, Oaxaca, Mexico -1,
Hughes CE 894, 47/87 -San Martín Pachivia, Guerrero, Mexico -10,
Hughes CE 903, 48/87 -Michoacán, Mexico -8. L. greggii S. Watson,
Hughes CE 695, 19/86 -El Barrial, Nuevo León, Mexico -6. L. lanceolata
S. Watson, Hughes CE 603, 44/85 -Escuinapa, Sinaloa, Mexico -7,
Hughes CE 631, 46/85 -Playa Azul, Michoacán, Mexico -10, Hughes
CE 1577, -Alamos, Sonora, Mexico -1. L. lempirana C.E. Hughes,
Hughes CE 1447, 5/91 -Valle del Aguán, Yoro, Honduras -16, Hughes
CE 1411, 6/91 -Cuyamapa, Yoro, Honduras -9. L. macrophylla subsp.
macrophylla Bentham, Hughes CE 2076, -Coxcatlan, Puebla, Mexico -1,
Hughes CE 1179, 55/88 -Vallecitos, Guerrero, Mexico -10, Hughes CE
2156, -Grutas de Cacahuamilca, Taxco, Guerrero, Mexico -1, Hughes CE
2164, -Cerro El Encinal, Iguala, Guerrero, Mexico -1. L. macrophylla
subsp. istmensis C.E. Hughes, Hughes CE 580, 47/85 -San Isidro Llano
Grande, Oaxaca, Mexico -10. L. magnifica (C.E Hughes) C.E. Hughes,
Hughes CE 412, 19/84 -El Rincón, Chiquimula, Guatemala -10, Hughes
CE 1089, 58/88 -El Carrizal, Chiquimula, Guatemala -8. L. matudae
(S. Zárate) C.E. Hughes, Hughes CE 2153, -San Miguel Tecuixiapan,
Guerrero, Mexico -1, Hughes CE 879, 49/87 -Mezcala, Guerrero,
Mexico -9, Hughes CE 2148, -San Juan Tetelcingo, Guerrero, Mexico
-1. L. multicapitula Schery, Hughes CE 1025, 81/87 -Los Santos,
Panama -13. L. pueblana Britton & Rose, Hughes CE 1648, 125/92
-Tehuacan Valley, Oaxaca, Mexico -1, Hughes CE 2089, -Cuicatlan,
Oaxaca, Mexico -1, Hughes CE 2140, -Santo Domingo Tonala, Oaxaca,
Mexico -1, Hughes CE 2077, -Teotitlán del Camino, Oaxaca, Mexico -1,
December 2011]
Govindarajulu et al.—Cryptic diversity and allopatry
Hughes CE 2092, -Dominguillo, Oaxaca,Mexico -1, Hughes CE
2139, -Santo Domingo Tonala, Oaxaca, Mexico -1. L. pulverulenta
(Schlechtendal) Bentham, Hughes CE 1051, 83/87 -Tamaulipas,
Mexico -4, Hughes CE 1058, 84/87 -Texas, USA -3 L. retusa Bentham
in Gray, Bendeck s.n., 23/86 -Coahuila, Mexico -9. L. salvadorensis
Standley ex Britton & Rose, Hughes CE 742, 17/86 -La Garita,
Choluteca, Honduras -8, Hughes CE 746, 34/88 -Choluteca, Honduras
-9, Hughes CE 36/88, 36/88 -La Garita, Choluteca, Honduras -9,
Hughes CE 1407, 7/91 -Esteli, Nicaragua -9, Hughes CE 98/90, 98/90
-La Garela, Choluteca, Honduras -9, Hughes CE 1211, 99/90 -Namali,
Choluteca, Honduras -4. L. shannonii Donnell Smith, Hughes
CE 1417, 1/91 -Jutiapa, Guatemala -10, Hughes CE 1676, 135/92
-Chiapas, Mexico -1, Hughes CE 1714, 141/92 -Yoro, Honduras -1,
Hughes CE 1399, 2/91 -La Puerta, Chontales, Nicaragua -4, Hughes
CE 239, 22/83 -Comayagua, Honduras -9, Hughes CE 282, 26/84
-Comayagua, Honduras -1, Hughes CE 507, 53/87 -Campeche, Mexico
2063
-3, Hughes CE 2166, -El Zamorano, Francisco Morazan, Honduras -1.
L. trichandra (Zuccarini) Urban, Hughes CE 1654, 128/92 -Oaxaca,
Mexico -1, Hughes CE 1682, 137/92 -Chiapas, Mexico -1, Hughes CE
1421, 4/91 -Erandique, Lempira, Honduras -10, Hughes CE 1106, 53/88
-Los Guates, Guatemala -5, Hughes CE 1130, 54/88 -Huehuetenango,
Guatemala -1, Hughes CE 1708, -Erandique, Lempira, Honduras -1,
Hughes CE 1709, -Erandique, Lempira, Honduras -1, Hughes CE 1710,
-Erandique, Lempira, Honduras -1. L. trichodes (Jacquin) Bentham,
Hughes CE 775, 2/86 -Cuicas, Trujillo, Venezuela -9, Hughes CE 997,
61/88 -Manabi, Ecuador -11, Hughes CE 1418, 3/91 -Copan, Honduras
-2. L. zacapana (C.E. Hughes) R. Govindarajulu & C.E. Hughes,
Hughes CE 299, 18/84 -Puerto de Golpe, El Progreso, Guatemala -1,
Hughes CE 1120, 56/88 -Vallecitos, Zacapa, Guatemala -8, Hughes CE
299, 15/83 -Puerto de Golpe, El Progreso, Guatemala -10, Hughes CE
1096, 57/88 -El Carrizal, Chiquimula, Guatemala -13.