JOURNAL OF CRUSTACEAN BIOLOGY, 34(6), 773-781, 2014

PHYLOGEOGRAPHY OF THE SCALLOPED SPINY-LOBSTER PANULIRUS HOMARUSRUBELLUS IN THE SOUTHWEST INDIAN OCEAN

Maggie M. Reddy 1,2, Angus H. H. Macdonald 1,∗,Johan C. Groeneveld 2, and Michael H. Schleyer 2

1 School of Life Sciences, University of KwaZulu-Natal, Westville Campus, Durban 4000, P. Bag X 54001, South Africa2 Oceanographic Research Institute, PO Box 10712, Marine Parade, Durban 4056, South Africa

A B S T R A C T

The Mozambique Channel between south-east Africa and Madagascar potentially forms a barrier to larval dispersal and thus gene flowacross the channel, even in species with long larval lifespans such as spiny lobsters. The DNA of 181 specimens of Panulirus homarusrubellus Berry, 1974, collected from six sites along the African coast and a site in Madagascar, was sequenced (591 base pairs) to investigategene flow along the coast and across the channel. Bayesian methods were implemented to infer genetic structure and relatedness. A total of112 haplotypes were recovered, of which 78% were unique. Samples from Africa and Madagascar did not share any haplotypes and differedby 78 mutations and 3% DNA divergence. These distinct lineages suggest an absence of contemporary gene flow across the MozambiqueChannel. Shallow genetic structure along approximately 1000 km of African coastline comprised combinations of seven distinct lineagesbut did not correspond to known biogeographic provinces. The African lineage appeared to be ancestral, and the source population for theMadagascan clade. Genetic isolation in refuges during glacial periods, followed by subsequent range expansions and secondary contact,may account for the high diversity in the African lineages.

KEY WORDS: connectivity, Island Biogeography Theory, larval dispersal, lineages, Mozambique Channel,Panulirus homarus rubellus

DOI: 10.1163/1937240X-00002284

INTRODUCTION

The scalloped spiny lobster Panulirus homarus (Linnaeus,1758) is widely distributed in the Indo-West Pacific, fromeastern South Africa to Somalia, Madagascar, along theArabian Peninsula, and from India and Indonesia to Japanand Australia (Holthuis, 1991). Three subspecies (or vari-eties) have been suggested, based on geographic distribu-tion and differences in sculpture on the pleon and colour(Berry, 1974a; Holthuis, 1991). The nominotypical P. h.homarus (Linnaeus, 1758) (green, microsculpta form) oc-curs throughout the range of the species, whereas P. h.megasculptus Pesta, 1915 (red, northern megasculpta form)occurs off Somalia and in the Arabian Sea. P. h. rubellus(red, southern megasculpta form) is restricted to the coastalwaters of eastern South Africa, southern Mozambique andsouth-east Madagascar (Berry, 1974a; Holthuis, 1991). Lav-ery et al. (2014) found that P. h. rubellus comprised the mostdivergent subspecies, and that it was monophyletic with re-spect to all other subspecies of P. homarus.

Panulirus h. rubellus inhabits shallow reefs from the surfzone to about 36 m depth, but is most common between 1-5 m (Berry, 1971a). It prefers turbid water and a temperaturerange of 17-24°C. It is active during the night, and inhabitscracks, crevices and reef overhangs during daylight, oftengregariously. P. h. rubellus reaches maturity after 2-3 years(50-60 mm carapace length), adults moult several timesper year, and egg-bearing occurs mainly during summer

∗ Corresponding author; e-mail: macdonalda@ukzn.ac.za

(Berry, 1971b). Large females can bear eggs up to fourtimes per year, and brood size increases steeply with lobstersize. Sub-adults often move short distances to deeper reefs,where most egg-bearing females are found, but no long-shore migrations have been observed in P. h. rubellus (Berry,1971a; Steyn and Schleyer, 2011).

Berry (1974b) collected 349 phyllosoma larvae of P. h.rubellus by night-time trawling with a small-mesh surfacenet off eastern South Africa. Most larvae were found within65 km of the coast, in inshore waters over the narrowcontinental shelf, and in the waters of the strong AgulhasCurrent. Larval duration was estimated to be at least 4months. These characteristics conform with the typicalpattern of spiny lobsters (Palinuridae), in which driftinglarvae may spend many months in the open ocean wherethey are dispersed by ocean currents (Booth and Phillips,1994; Cobb, 1997). Given the high potential for gene flowin ocean currents, most gene flow models assume genetichomogeneity among individuals, unless they are subjectedto genetic isolation through vicariance or dispersal barriers(Avise et al., 1987; Thorpe et al., 2000).

The present day the southwest (SW) Indian Ocean is adynamic region influenced by large scale ocean currentssuch as the Agulhas Current, East Madagascar Current andthe Mozambique Channel eddies (Fig. 1). These featuresmay facilitate wide dispersal of lobster larvae (Tolley etal., 2005) or form hydrological barriers that restrict larval

© The Crustacean Society, 2014. Published by Brill NV, Leiden DOI:10.1163/1937240X-00002284

774 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 34, NO. 6, 2014

Fig. 1. Study area showing the collection sites for Panulirus homarus rubellus specimens in southern Mozambique, eastern South Africa and south-eastern Madagascar. The relevant ocean current and eddy systems are indicated, after Lutjeharms (2006). Haplotype frequencies are shown for lobsterscollected at each of the seven sites. This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/1937240x.

dispersal (Gopal et al., 2006). For instance, it is yet unclearwhether lobster larvae are able to disperse across theMozambique Channel between Madagascar and the Africancoast, or whether the Mozambique Channel forms a geneticbarrier to cross-channel dispersal, despite long larval lifespans. Furthermore, at least two coastal phylogeographicbreaks are recognized in eastern South Africa, betweenwarm temperate and subtropical biogeographic provinces(Algoa Bay to Wild Coast), and between subtropical andtropical provinces near the Mozambique border (Teske etal., 2011). Several coastal species are genetically structuredacross these biogeographic disjunctions, with some dataindicating the pattern of population division is driven by bothdispersal mode and ecological drivers (Teske et al., 2011).

In the late Pleistocene, successive glaciations and sea levelchanges occurred, with the last glaciation event peakingapproximately 19,000-22,000 years ago (Yokoyama et al.,2000). The population expansion dates of several spinylobster species in the SW Indian Ocean and South Atlanticcorrelate well with this period (Tolley et al., 2005; Gopal etal., 2006; von der Heyden et al., 2007), when changes in sealevel, water temperature and ocean current patterns wouldhave influenced species distribution and larval dispersalpatterns.

Mitochondrial DNA (mtDNA) has often been used toanalyse the genetic structure of spiny lobster populations(Brasher et al., 1992; Ovenden et al., 1992, 1997; Palero etal., 2008; Chow et al., 2012). Farhadi et al. (2013) showedgenetic differentiation in mtDNA control region sequencesin P. h. megasculptus from the Northwest Indian Ocean; inthe SW Indian Ocean, shallow structure occurred in Palin-

urus delagoae from Mozambique and eastern South Africa(Gopal et al., 2006). Palinurus gilchristi Stebbing, 1990 insouthern South Africa (Tolley et al., 2005) and Palinurusbarbarae Groeneveld, Griffiths, and Van Dalsen, 2006 onsubmerged seamounts south of Madagascar were panmictic(pers. com. C. A. Matthee, University of Stellenbosch). Ja-sus paulensis (Heller, 1862) at remote islands and seamountsin South Atlantic and SW Indian Ocean waters showed highconnectivity and apparently no barriers to gene flow overthousands of kilometres (Groeneveld et al., 2012). In otheroceans, genetic structure has been found in Palinurus ele-phas (Fabricius, 1787) from the Northeast (NE) Atlantic andMediterranean (Palero et al., 2008), and Panulirus penicilla-tus (Olivier, 1791) from the East and Central/Western Pacific(Chow et al., 2012). MtDNA analysis did not differentiateJasus edwardsii (Hutton, 1875) from across the Tasman Sea(Ovenden et al., 1992, 1997) but recent studies using mi-crosatellites distinguished low levels of population differen-tiation between Australia and New Zealand (Morgan et al.,2013; Thomas and Bell, 2013).

We analysed cytochrome c oxidase subunit I-like DNA(COI-like) to test the hypothesis that there is no geneticstructure amongst P. h. rubellus indivduals in the SW In-dian Ocean. The analysis included samples from the Africanmainland (Mozambique and eastern South Africa) andsouth-east Madagascar, to investigate whether the Mozam-bique Channel currents form an oceanographic barrier togene flow. The results were interpreted within the contextof past climate and geological events, and likely larval dis-persal patterns.

REDDY ET AL.: PHYLOGEOGRAPHY OF PANULIRUS HOMARUS IN THE SW INDIAN OCEAN 775

MATERIALS AND METHODS

Lobsters were collected by SCUBA and snorkelling or purchased from fish-ers at four sites in eastern South Africa, two sites in southern Mozambique,and a site in south-eastern Madagascar (Fig. 1). The carapace length and sexof each lobster was recorded and a picture taken as a voucher for identifi-cation. Subspecies identification followed the physical description by Berry(1974a). A leg segment was excised from each lobster, preserved in 70%ethanol, transported on ice and stored at 4°C for later DNA isolation.

Genomic DNA was isolated from approximately 50 mg of tissue. Thestandard protocol for the ZYMO ZR96 tissue and insect DNA kit™ wasmodified by adding 20 μl of proteinase K (Fermentas, Inqaba BiotechnicalIndustry) and incubating overnight at 56°C. The method recovered between20 and 30 ng μl−1 DNA.

The COI-like region was PCR-amplified (Saiki et al., 1988) usingprimers published in Chow et al. (2006) for identification of individualPanulirus. The primers were optimised for the amplification of the regionfor P. h. rubellus. PCR was carried out in a final volume of 40 μl comprisedof 21 μl PCR water, 4 μl 10× buffer (Super-Therm®, Industricord), 0.18 μl25 mM MgCl2, 0.15 μl 10 mM deoxynucleoside-triphosphate mixture(dNTP mix), 0.84 μl 10 mM forward primer, 0.84 μl 10 mM reverse primer,0.2 μl 5 μl−1 Taq polymerase (Super-Therm®, Industricord) and 10 μl30 ng μl−1 template DNA. The thermo-cycling conditions were 5 min at70°C, 94°C for 30 s, 60°C for 30 s and 72°C for 1 min; steps 2-4 wererepeated 29 times, followed by a final extension at 72°C for 10 min. ThePCR products were sequenced using an ABI 3730 capillary sequencer andBigEnd Dye chemistry.

Sequences were edited in BioEdit v. 7.0.9.0. (Hall, 1999) and alignedusing CLUSTAL X (Thompson et al., 1994). The entire data set wasconverted into amino acids using MEGA v. 4 (Kumar et al., 2009) to checkfor stop codons in the reading frame of the sequences.

Sequences were tested for substitution saturation using DAMBE soft-ware (Xia and Lemey, 2009). A haplotype list was generated in DnaSPv. 5.10 (Librado and Rozas, 2009) and nucleotide and haplotype diver-sity were calculated per site, region and all data. Sequence divergence be-tween sites was calculated in DnaSP v. 5.10. Mismatch distribution statis-tics, Theta (θ)-effective population size (Tajima, 1993), Tau (T ) and good-ness of fit tests: Sum of squares deviation (SSD), Harpendings RaggednessIndex (HRI), Probabilities (p) were calculated using Arlequin v. 3.5 (Ex-coffier and Lischer, 2010). Tajima’s D (Tajima, 1989) was used to test se-lective neutrality and Fu’s Fs (Fu, 1997) to test mutation drift equilibriumfrom which recent demographic changes could be inferred. Although pri-marily designed to test the selective neutrality of markers, these tests haverecently been used to make inferences about population sizes and expan-sion (Silva et al., 2010). Harpending’s Raggedness Index (HRI) was used totest for population expansion in models of demographic change and spatialexpansion (Rogers and Harpending, 1992).

An AMOVA was run in GenAlEx v. 6.4 (Peakall and Smouse, 2006)using 1000 random permutations to calculate the confidence intervals forpartitioning between groupings. In the present study, the data representedhaploid-DNA and, therefore, a �PT analogue of Fst was used (McMillen-Jackson and Bert, 2004). In addition, �PR was used to calculate the genetic

difference between sites within a region and �RT the genetic differencebetween regions. Pairwise �PT values between populations were alsocalculated.

Phylogeographic lineages were detected using the molecular softwareprogram BAPS v. 6 (Corander et al., 2013). Individuals were assigned todifferent genetic groups using a Bayesian approach. This method reducedthe bias common in AMOVA analysis that requires the pre-assigning ofgroups based on spatial information or other a priori assumptions. A seriesof genetic clustering analyses were run for different numbers of populations(k). Default settings were used for all BAPS analyses.

A phylogenetic tree was constructed using MrBayes and the general-time reversible model of evolution with a gamma distribution of 0.27,generated in Mr Modeltest (Tavare, 1986; Ronquist and Huelsenbeck, 2003;Nylander, 2004). No specific root was designated as no outgroup could beused as the sequences here are numts and not COI sequences.

RESULTS

A total of 181 P. h. rubellus samples were successfullysequenced for the COI-like region. Sequences were trimmedto 591 base pairs, of which 538 were constant and 52were parsimony informative. Stop codons and indels werediscovered in all sequences and thus, the sequences werepossibly from a pseudogene. Xia’s test in DAMBE indicatedthat there was little substitution saturation. Base frequenciesfor the alignment of the entire dataset were A = 26.5%, T =34.5%, G = 15.5% and C = 23.5%.

A total of 112 haplotypes were recovered with a totalhaplotype diversity of 0.98 ± 0.003. Some 78% of haplo-types were unique and deposited in GenBank (KJ671893-KJ672073). Haplotype (h) and nucleotide diversity (π)ranged between 0.97-1.00 and 0.0079-0.0208, respectively(Table 1). Samples from Africa and Madagascar did notshare any haplotypes, and all Madagascar haplotypes wereunique. The most frequently observed haplotype occurred in11 individuals.

High haplotype and low nucleotide diversity found at Chi-denguele and Blood Reef reflect expanding populations withan initial low effective population size (von der Heyden etal., 2008), whereas high haplotype and nucleotide diversityobserved at other sites correspond to larger original popula-tions.

Tajima’s D (p > 0.05) indicated that the COI-like mark-er conformed to selective neutrality (Table 2). Values werenegative for each site (except Mdumbi), implying a large ini-tial population size, and a large expansion event (McMillen-

Table 1. Intra-subspecies indices for Panulirus homarus rubellus from the SW Indian Ocean calculated per site, region and all data as from partial COI-likesequence data. N , sample size; S, number of polymorphic sites; M , total number of mutations; K , nucleotide differences; π , nucleotide diversity; H , numberof haplotype; h, haplotype diversity; SD, standard deviation.

Site Latitude (S) Longitude (E) N S M K π H h

Chidenguele 34.21280 24.95556 30 27 30 4.664 0.0079 24 0.9839Xai Xai 33.73417 25.11889 25 51 56 12.307 0.0208 22 0.9867Blood Reef 31.05833 29.88278 25 28 34 5.773 0.0098 23 0.9933Scottburgh 30.76056 30.28917 42 33 38 5.914 0.0100 32 0.9826Port St Johns 29.54917 31.63889 21 50 55 11.709 0.0198 20 0.9947Mdumbi 29.21887 31.93042 23 38 42 11.731 0.0199 18 0.9760Fort Dauphin 25.03330 46.98330 15 36 39 10.886 0.0184 15 1.000

Africa 166 53 62 8.647 0.0146 97 0.985Madagascar 15 36 39 10.886 0.0184 15 1.000

Total 181 67 78 10.065 0.0170 112 0.9880

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Table 2. Neutrality tests, Tajima’s D and Fu’s Fs scores for a fragmentof the COI-like gene in Panulirus homarus rubellus from the SW IndianOcean, with corresponding p values for each sampling site, region (Africaand Madagascar) and all data.

Site Tajima’s D P Fu’s Fs p

Chidenguele −1.131 0.142 −18.521 0.000Xai Xai −0.342 0.402 −8.056 0.007Blood Reef −0.824 0.215 −18.454 0.000Scottburgh −0.788 0.247 −23.724 0.000Port St Johns −0.628 0.281 −9.142 0.001Mdumbi 0.538 0.753 −3.880 0.085Fort Dauphin −0.071 0.500 −6.985 0.005

Africa −0.219 0.487 −24.444 0.000Madagascar −0.071 0.500 −6.985 0.005

Total −0.460 0.434 −24.228 0.001

Jackson and Bert, 2004). Selective neutrality was fur-ther supported by high levels of genetic diversity (García-Merchán et al., 2011). Fu’s Fs (p < 0.05) indicated a de-parture from mutation-drift equilibrium. HRI was not sig-nificant (Table 3), which supports the hypothesis of recentspatial expansion and these results concur with Fu’s Fs.

AMOVAs were run to test the partitioning of genetic vari-ation between sites. The regional grouping that resulted inthe highest percentage of genetic variation was a delineationbetween Africa, Madagascar and a lineage containing spec-imens that superficially resembled P. h. homarus or were in-termediate in colour and pleonal sculptural pattern betweenP. h. homarus and P. h. rubellus but were genetically similarto P. h. rubellus. This revealed a high level of differentiationbetween regions (p = 0.001; �PT = 0.626) but not betweenpopulations within regions (p = 0.005; �PR = 0.041). Nev-ertheless, a large proportion (37%) of genetic variation oc-curred within populations.

Pairwise �PT was calculated between site pairs and valuesranged from 0.01-0.563 (Table 4). The highest differentia-tion along the African coast was found between Chidengueleand Scottburgh (�PT = 0.403), followed by Xai Xai andPort St Johns (�PT = 0.370). The Blood Reef and Xai Xai

Table 3. Results of mismatch distribution analysis testing in a spatialexpansion model assuming a constant deme size, associated goodness offit and corresponding p values, tested on a fragment of the COI-likegene of Panulirus homarus rubellus from the SW Indian Ocean. θ , theta(effective population size) initial; T , tau; SSD, sum of squares deviation;HRI, Harpendings Raggedness Index.

Site θ T SSD p HRI p

Chidenguele 0.004 4.997 0.0027 0.560 0.0166 0.680Xai Xai 11.264 35.73 0.0420 0.210 0.0181 0.860Blood Reef 0.007 3.868 0.0064 0.130 0.0242 0.580Scottburgh 1.132 4.679 0.0011 0.850 0.0108 0.810Port St Johns 7.101 2.424 0.0866 0.600 0.0115 0.730Mdumbi 14.183 1.487 0.0131 0.350 0.0132 0.680Fort Dauphin 5.281 5.401 0.0173 0.260 0.0350 0.190

Africa 5.281 5.401 0.0173 0.260 0.0350 0.190Madagascar 3.340 2.867 0.0054 0.410 0.0074 0.700

Total 6.361 2.307 0.0056 0.600 0.0053 0.740

sites were both genetically differentiated from all other sites(Table 4, above diagonal). Madagascar was significantly dif-ferentiated from all other sites (�PT = 0.5). The highestoverall level of genetic differentiation was found betweenChidenguele (Mozambique) and Fort Dauphin (Madagas-car).

DNA sequence divergence revealed marked differencesbetween sites, particularly between Africa and Madagascar(78 mutations and 3% DNA divergence) (Table 5). Sequencedivergence was highest (3.4%) between specimens fromMdumbi (Africa) and Fort Dauphin (Madagascar; Table 5).Chidenguele yielded the lowest level of differentiation fromBlood Reef and Scottburgh.

The admixture plot was constructed using individualsamples which were assigned a different colour accordingto genetic lineage, regardless of spatial information (Fig. 2).Six lineages were recovered, with a single lineage prolificalong the African coast (blue) and another along Madagascar(green). Two individuals in the Madagascan lineage sharedan ancestral source with an African lineage, and three fromAfrica could not be assigned to a single lineage. The spatialgenetic mixture plot was generated using genetic and apriori spatial information for individual samples (Fig. 3),and the distribution of lineages was similar to that of theadmixture plot.

In the phylogram, the Madagascan lineage was well-supported (posterior probability of 1) and distinct from theAfrican lineage (Fig. 4). This is supported by the resultsobtained from the BAPS and AMOVA analyses whichindicate that the Madagascan specimens belong to a lineagethat is isolated from south-east Africa. In addition, a cladewith well-supported internal nodes (posterior probability of1) consisted of specimens that superficially resembled P.h. homarus or were intermediate in colour and abdominalsculptural pattern between P. h. homarus and P. h. rubellusand this was supported in previous analyses using AMOVA.

DISCUSSION

To verify that the sequences obtained were from the COI re-gion of P. h. rubellus, we used BLAST (Basic Local Align-ment Search Tool) to search the NCBI (National Centre forBiotechnology Information) nucleotide database for simi-lar sequences (Altschul et al., 1990). All searches showedour sequences to be similar to those deposited on Gen-Bank by other researchers, and catalogued as P. homarusmitochondrial COI region with high e-values, indicative offew mismatching nucleotides. None-the-less, upon deposit-ing the sequences in the NCBI repository and using the spe-cial mtDNA codons for crustaceans, it became apparent thatthe sequences were nuclear copies of the mtDNA COI regioninserted into the genome of P. h. rubellus. These sequences(known as numts) are common in crustacean DNA, and canaffect the interpretation of data (Buhay, 2009) depending onthe research question. Importantly, nuclear DNA has slowersubstitution and mutation rates than mtDNA in crustaceans,and therefore reflects deeper divergences between lineagesthan mtDNA (Zhang and Hewitt, 1996). The sequence datagenerated in this study reflects an ancestral state of the P. h.rubellus mitochondrion and may only be reliably comparedto other numts (Bensasson et al., 2001). For this reason we

REDDY ET AL.: PHYLOGEOGRAPHY OF PANULIRUS HOMARUS IN THE SW INDIAN OCEAN 777

Table 4. Pairwise �PT site-comparison for Panulirus homarus rubellus in the SW Indian Ocean; p values are presented above the diagonal with �PTvalues below the diagonal. Pairwise comparisons were based on 1000 permutations. ∗Significant values.

Site Chidenguele Xai Xai Blood Reef Scottburgh Port St Johns Mdumbi Fort Dauphin

Chidenguele – 0.030 0.280 0.440 0.010 0.010 0.010Xai Xai 0.056 – 0.330 0.030 0.400 0.060 0.010Blood Reef 0.013 0.008 – 0.170 0.220 0.010 0.010Scottburgh 0.000 0.044∗ 0.013 – 0.010 0.010 0.010Port St Johns 0.081∗ 0.000 0.013 0.059∗ – 0.140 0.010Mdumbi 0.244∗ 0.052 0.144∗ 0.217∗ 0.026 – 0.010Fort Dauphin 0.563∗ 0.404∗ 0.516∗ 0.542∗ 0.410∗ 0.435∗ –

Table 5. DNA sequence divergence indicated by the number of shared mutations (above) and the average nucleotide substitution per site (%) and betweensampling localities (below) for Panulirus homarus rubellus in the SW Indian Ocean.

Chidenguele Xai Xai Blood Reef Scottburgh Port St Johns Mdumbi Fort Dauphin

Chidenguele – 27 19 22 24 24 18Xai Xai 1.5% – 30 36 51 39 28Blood Reef 0.1% 1.5% – 29 31 30 24Scottburgh 0.1% 1.6% 1.0% – 36 32 26Port St Johns 1.5% 2.0% 1.5% 1.6% – 38 28Mdumbi 1.8% 2.1% 1.7% 1.8% 2.0% – 25Fort Dauphin 2.7% 3.3% 2.7% 2.8% 1.3% 3.4% –

Fig. 2. Admixture plot (Corander et al., 2006) generated in BAPS v. 6 based on 591 bp of the COI-like gene for 181 individuals. Each bar representsindividual samples which were assigned different colours according to the genetic lineage to which they belong. The African (blue) and Madagascar (green)regions were each dominated by a single lineage.

Fig. 3. Spatial genetic mixture analysis (Corander et al., 2008; Cheng et al., 2013) constructed from partial COI-like sequence data, implemented usingBAPS v. 6. Three genetic (blue, orange, green) lineages are prolific along the African coast and show no genetic mixing between lineages, while two lineagesoccur in Madagascar (orange and green), one of which is shared with Africa (orange).

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Fig. 4. The Bayesian phylogram generated using Mr Bayes provided statistical support for the monophyly of Panulirus homarus rubellus. Thedistinct Madagascan clade (green) appeared to be derived from the major African clade (blue). Only posterior probabilities of 1 for nodal support areindicated in the phylogram. This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/1937240x.

did not attempt to estimate population expansion dates usingpublished mtDNA mutation rates.

The DNA results revealed significant genetic partitioningbetween P. h. rubellus in south-east Madagascar and thosefrom the African shelf, and the absence of shared haplotypessuggests that little contemporary gene flow occurs across theMozambique Channel for this lobster. Restricted gene flowacross and within the channel has also been found in in-tertidal barnacles in Tetraclita Schumacher, 1817 (Tsang et

al., 2012), crown-of-thorn starfish Acanthaster planci (Lin-naeus, 1758) (Volger et al., 2012), several ophiuroid brittle-stars (Hoareau et al., 2013), and the reef fish Myripristisberndti (Jordan and Everman, 1903) (Muths et al., 2011).Restricted gene flow despite all of these species havingpelagic early life history phases, with larvae or eggs that maydisperse widely in ocean currents suggest that the Mozam-bique Channel (approx. 430 km at the narrowest point) actsas a genetic barrier in the SWIO region. Given the poten-

REDDY ET AL.: PHYLOGEOGRAPHY OF PANULIRUS HOMARUS IN THE SW INDIAN OCEAN 779

tial for larval transport across the Mozambique Channel viaeddies larval behaviour may act to retain larvae or returnthem to natal areas (Chiswell and Booth, 1999; Butler et al.,2011), or alternatively, propagules that reach the oppositeside of the channel may be subject to selection (Warner andCowen, 2002).

The African and Madagascan lineages of P. h. rubellusare monophyletic, confirming that they belong to a singlesubspecies or possibly species (data unpublished; Lavery etal., 2014). The relationship indicates that the two lineagesonce shared a common ancestor, and the derived Madagas-can clade suggests an African origin, consistent with the is-land biogeography theory which suggests that most islandsare colonised from mainland source populations (MacArthurand Wilson, 1967; Warren et al., 2010). However, distinctlineages have formed probably due to restricted gene flowacross the Mozambique Channel. These findings could rep-resent an early stage of peripatric speciation, where distinctlineages form as a prelude to speciation, given sufficienttime and a sustained interruption in gene flow. It is worthnoting, the African and Madagascan lineages may currentlyrepresent cryptic species or subspecies which are geneticallydistinct, but have not yet evolved morphologically distincttraits. The high genetic divergence between the African andMadagascan lineages (3%) is similar to that found betweenthe recognised subspecies P. h. rubellus and P. h. homarus(unpublished data; Lavery et al., 2014).

A genetically distinct African clade compromised of indi-viduals that superficially resembled P. h. homarus or wereintermediate in colour and pleonal sculptural pattern be-tween P. h. homarus and P. h. rubellus but, were geneticallymore closely related to P. h. rubellus. This pattern suggestedsome level of hybridisation occurs between P. h. homarusand P. h. rubellus. However, further investigation using fasterevolving nuclear markers are needed to support the hybridi-sation hypothesis.

Given that the approximately 20 species of Panulirus mostprobably evolved from a common Pacific ancestor withinthe past ca. 5 million years (George and Main, 1967; Pol-lock, 1992), the much earlier breakup of Gondwanaland(Jurassic) and drift of Madagascar to its present position(early Cretaceous) cannot account for the genetic structureobserved in P. h. rubellus. The alternative explanation isthat colonization events occurred through larval dispersaland exchange, followed by isolation resulting from sea levelchanges during glacial periods (Pollock, 1992). Sea levelduring glacial was periods was lower than at present (Lam-beck et al., 2002) and, under these circumstances, palaeo-oceanographic model simulations support larval transporthypotheses of Madagascan P. h. rubellus from an Africansource in the Miocene (see Ali and Huber, 2010; Krause,2010). This is consistent with the predominantly Africanorigin of most present-day biota of Madagascar (Yoder andNowak, 2006).

Higher sea levels and stronger currents, in particular thepresent-day flow direction and strength of the Agulhas Cur-rent which formed during the Miocene-Pliocene boundary(5-3 MYA; Martins et al., 1982), appear to disrupt con-temporary larval exchange and gene flow between Mada-gascar and Africa, despite the apparent favourable trans-

port of water masses across the channel in mesoscale anti-cyclonic eddies (Lutjeharms, 2006). These eddies originatenear the coast of south-east Madagascar where the south-ern limb of the East Madagascar Current terminates in aneastwards retroflection, and in occasional vortex dipoles thatcarry shelf waters from Madagascar westwards towards theAfrican shelf (de Ruijter et al., 2004). The hypothesis thatthese eddies would regularly transport long-lived lobster lar-vae across the channel from south-east Madagascar to theAfrican shelf, thus resulting in gene flow across the Mozam-bique channel, was rejected by this study.

Genetic structure along the African coast was also recov-ered, but was shallow. The structure along the African coastdid not correspond to known biogeographic provinces (seeTeske et al., 2011), and instead, the six sampled populationsalong the approximately 1000 km coastline were made upof combinations of seven distinct lineages (Fig. 2). Theselineages may have formed during glacial activity when theybecame fragmented and isolated in refuges along the coast(see also Tolley et al., 2005; Panova et al., 2011; Teske etal., 2011). The accumulation of mutations in these isolatedpopulations over time presumably gave rise to geneticallydistinct lineages, which subsequently intermixed along thecoast with the rise in sea-level and strengthening of cur-rents following the LGM. This theory is supported by thehigh genetic diversity, large percentage of unique haplo-types, MMDs and Fu’s Fs.

The African lineages of P. h. rubellus remain relativelydisconnected despite the potential for intermittent contem-porary gene flow along the coast facilitated by eddies andcounter currents as well as Natal pulses along the near shoreedge of the Agulhas Current. The northern Agulhas Currentclosely follows the shelf-edge of southern Mozambique andeastern South Africa (Lutjeharms, 2006), mostly within tensof kilometres from the shore. Assuming that larvae originat-ing from the African coast are lost if they are caught up inthe Agulhas Current core (i.e., as soon as they cross the shelfedge), then successful dispersal and settlement would be re-stricted to near shore waters.

Spiny lobster larvae are active swimmers and may usevertical positioning in the water column to remain close tothe coast or return shoreward (reviewed by Phillips et al.,2006; Butler et al., 2011). Onshore movements may involvea combination of active swimming and the use of naturalonshore advection or passive processes, such as wind-drivensurface currents. Larval retention in shelf waters inshoreof the Agulhas Current core, and their return to the coast,can therefore be facilitated by persistent eddies in the NatalBight and near Durban, and counter-currents north and southof the Bight (see Fig. 1). These are likely mechanismsthat may maintain a degree of genetic structure in lobsterpopulations between Chidenguele and Mdumbi, despite longlarval life spans.

From an evolutionary perspective, larval retention closeto natal grounds may ensure a higher recruitment successto suitable environments (Toonen and Pawlik, 2001; Strath-mann et al., 2002). Local larval retention and habitat speci-ficity have been documented for other spiny lobster species(Iacchei et al., 2013) and appear to be more prevalentamong marine organisms than previously thought (Warner

780 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 34, NO. 6, 2014

and Cowen, 2002; Ayre et al., 2009; Yednock and Neigel,2011; Mullaney and Suthers, 2013).

In conclusion, P. h. rubellus comprises a monophyleticclade within its known distribution range in south-eastAfrica and Madagascar, and its origin appears to be African,with subsequent dispersal to Madagascar. The distinct Af-rican and Madagascan lineages refute earlier hypotheses ofcontemporary, regular gene flow across the MozambiqueChannel (Berry, 1974a), despite a long larval duration andapparently favourable flow of mesoscale eddies betweenMadagascar and south-east Africa. Several phylogeographiclineages on the African shelf may have formed due to geneticisolation during recent glaciations, and subsequent rangeexpansion and secondary contact may partly account for thehigh diversity of COI-like DNA.

ACKNOWLEDGEMENTS

We thank the South West Indian Ocean Fisheries Project (SWIOFP) and theAfrican Coelacanth Ecosystem Programme (ACEP) Phase III for fundingthis study. The South African Association for Marine Biological Research(SAAMBR) provided office and laboratory facilities. Erika Steyn and JadeMaggs assisted with the collection of lobsters.

REFERENCES

Ali, J. R., and M. Huber. 2010. Mammalian biodiversity on Madagascarcontrolled by ocean currents. Nature 463: 653-656.

Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.Basic local alignment search tool. Journal of Molecular Biology 215:403-410.

Avise, J., J. C. Arnold, R. M. Ball, E. Bermingham, T. Lamb, J. E. Neigel,C. A. Reeb, and N. C. Saunders. 1987. Intraspecific phylogeography:the mtDNA bridge between population genetics and systematics. AnnualReview of Ecology and Systematics 18: 489-522.

Ayre, D. J., T. E. Minchinton, and C. Perrin. 2009. Does life history predictpast and current connectivity for rocky intertidal invertebrates across amarine biogeographic barrier? Molecular Ecology 18: 1887-1903.

Bensasson, D., D. Zhang, D. L. Hartl, and G. M. Hewitt. 2001. Mitochon-drial pseudogenes: evolution’s misplaced witnesses. Trends in Ecologyand Evolution 16: 314-321.

Berry, P. F. 1971a. The spiny lobsters (Palinuridae) of the coast of southernAfrica: distribution and ecological notes, pp. 4-23. In, South AfricanAssociation for Marine Biological Research, Investigational Reportno. 27.

. 1971b. The biology of the spiny lobster Panulirus homarus(Linnaeus) off the east coast of southern Africa, pp. 3-75. In, SouthAfrican Association for Marine Biological Research, InvestigationalReport no. 28. South African Association of Marine Biological Research,Durban.

. 1974a. A revision of the Panulirus homarus-group of spiny lobsters(Decapoda, Palinuridae). Crustaceana 27: 31-42.

. 1974b. Palinurid and scyllarid lobster larvae of the Natal coast,South Africa, pp. 3-44. In, South African Association of Marine Biolog-ical Research, Investigational Report no. 34. South African Associationof Marine Biological Research, Durban.

Booth, J. D., and B. F. Phillips. 1994. Early life history of spiny lobster.Crustaceana 66: 271-294.

Brasher, D. J., J. R. Ovenden, and R. W. G. White. 1992. MitochondrialDNA variation and phylogenetic relationships of Jasus spp. (Decopoda:Palinuridae). Journal of Zoology (London) 227: 1-16.

Buhay, J. E. 2009. “COI-like” sequences are becoming problematic inmolecular systematic and DNA barcoding studies. Journal of CrustaceanBiology 29: 96-110.

Butler IV, M. J., C. B. Paris, J. S. Goldstein, H. Matsuda, and R. K.Cowen. 2011. Behaviour constrains the dispersal of long-lived spinylobster larvae. Marine Ecology Progress Series 422: 223-237.

Cheng, L., T. R. Connor, J. Sirén, D. M. Aanensen, and J. Corander. 2013.Hierarchical and spatially explicit clustering of DNA sequences withBAPS software. Molecular Biology and Evolution 30(5): 1224-1228.

Chiswell, S. M., and J. D. Booth. 1999. Rock lobster Jasus edwardsiilarval retention by the Wairarapa Eddy off New Zealand. Marine EcologyProgress Series 183: 227-240.

Chow, S., A. Jeff, Y. Miyake, K. Konishi, M. Okazaki, N. Suzuki, M. F.Abdullah, H. Imai, T. Wakabayasi, and M. Sakai. 2012. Genetic isolationbetween western and eastern Pacific populations of pronghorn spinylobster Panulirus penicillatus. PLoS One 6: e29280.

, N. Suzuki, H. Imai, and T. Yoshimura. 2006. Molecular speciesidentification of spiny lobster phyllosoma larvae of the genus Panulirusfrom the Northwestern Pacific. Marine Biotechnology 8: 260-267.

Cobb, J. S. 1997. Oceanic processes affecting lobster larvae: report from aworkshop. Marine and Freshwater Research 48: 771-775.

Corander, J., and P. Marttinen. 2006. Bayesian identification of admixtureevents using multilocus molecular markers. Molecular Ecology 15: 2833-2843.

, J. Sirén, and E. Arjas. 2008. Bayesian spatial modelling of geneticpopulation structure. Computational Statistics 23: 111-129.

, L. Cheng, P. Marttinen, J. Sirén, and J. Tang. 2013. BAPS:Bayesian Analysis of Population Structure. Manual, Version, 6.

de Ruijter, P. M. W., H. M. van Aken, E. J. Beier, J. R. E. Lutjeharms,R. P. Matano, and M. W. Schouten. 2004. Eddies and dipoles aroundSouth Madagascar: formation, pathways and large-scale impact. DeepSea Research Part I: Oceanographic Research Papers 51: 383-400.

Excoffier, L., and H. E. Lischer. 2010. Arlequin suite ver 3.5: a new seriesof programs to perform population genetic analyse under Linux andWindows. Molecular Ecology Resources 10: 564-567.

Farhadi, A., H. Farhamand, M. A. Nematollahi, A. Jeffs, and S. D. Lavery.2013. Mitochondrial DNA population structure of the scalloped lobsterPanulirus homarus (Linnaeus 1758) from the West Indian Ocean. ICESJournal of Marine Science 70: 1491-1498.

Fu, Y. X. 1997. Statistical tests of neutrality of mutations against populationgrowth, hitchhiking and background selection. Genetics 147: 915-925.

García-Merchán, V. H., A. Robainas-Barcia, P. Abelló, F. Palero, M. García-Rodríguez, M. Gil de Sola, and M. Pascual. 2011. Phylogeographicpatterns of decapod crustaceans at the Atlantic-Mediterranean transition.Molecular Phylogenetics and Evolution 62: 664-672.

George, R. W., and A. R. Main. 1967. The evolution of spiny lobsters(Palinuridae): a study of evolution in the marine environment. Evolution21: 803-820.

Gopal, K., K. A. Tolley, J. C. Groeneveld, and C. A. Matthee. 2006. Mito-chondrial DNA variation in spiny lobster Palinurus delagoae suggestsgenetically structured populations in the south western Indian Ocean.Marine Ecology Progress Series 319: 191-198.

Groeneveld, J. C., S. von der Heyden, and C. A. Matthee. 2012. Highconnectivity and lack of mtDNA differentiation among two previouslyrecognized spiny lobster species in the southern Atlantic and IndianOceans. Marine Biology Research 8: 764-770.

Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignmenteditor and analysis program for Windows 95/98/NT. Nucleic AcidsSymposium Series 41: 95-98.

Hoareau, T. B., E. Boissin, G. Paulay, and J. H. Bruggemann. 2013.The Southwestern Indian Ocean as a potential marine evolutionaryhotspot: perspectives from comparative phylogeography of reef brittle-stars. Journal of Biogeography 40: 2167-2179.

Holthuis, L. B. 1991. FAO Species Catalogue. Marine lobsters of theworld. An annotated and illustrated catalogue of species of interest tofisheries known to date. FAO Fisheries Synopsis 125. Rome, Food andAgricultural Organization of the United Nations 13: 292.

Iacchei, M., T. Ben-Horin, K. A. Selkoe, C. E. Bird, F. J. García-Rodríguez, and R. T. Toonen. 2013. Combined analyses of kinship andFST suggest potential drivers of chaotic genetic patchiness in high gene-flow populations. Molecular Ecology 22: 3476-3494.

Krause, D. W. 2010. Biogeography: washed up in Madagascar. Nature 463:613-614.

Kumar, V., A. Dey, and A. Singh. 2009. MEGA: bio-computationalsoftware for sequences and phylogenetic analysis. Proceedings of theWorld Congress on Engineering 2: 1-3.

Lambeck, K., T. M. Esat, and E. K. Potter. 2002. Links between climateand sea levels for the past three million years. Nature 419: 199-206.

Lavery, S. D., A. Farhadi, H. Farahmand, T. Y. Chan, A. Azhdehakoshpour,V. Thakur, and A. G. Jeffs. 2014. Evolutionary divergence of geographicsubspecies within the scalloped spiny lobster Panulirus homarus (Lin-naeus 1758). PLoS One 9: e97247.

REDDY ET AL.: PHYLOGEOGRAPHY OF PANULIRUS HOMARUS IN THE SW INDIAN OCEAN 781

Librado, P., and J. Rozas. 2009. DnaSP v5: a software for comprehensiveanalysis of DNA polymorphism data. Bioinformatics 25: 1451-1452.

Linnaeus, C. 1758. Systema Naturae per Regna Tria Naturae, SecundumClasses, Ordines, Genera, Species, cum Characteribus, Differentiis,Synonymis, Locis. 10th Edition. Laurentius Salvius, Stockholm.

Lutjeharms, J. R. E. 2006. Three decades of research on the greater AgulhasCurrent. Ocean Science Discussions 3: 939-995.

MacArthur, R. H., and E. O. Wilson. 1967. The Theory of IslandBiogeography. Princeton University Press, Princeton, NJ.

Martins, A. K., S. W. Goodlad, and D. A. Salmon. 1982. Sedimentary basinin-fill in the northernmost Natal Valley, hiatus development and AgulhasCurrent palaeo-oceanography. Journal of the Geological Society 139:183-201.

McMillen-Jackson, A. L., and T. M. Bert. 2004. Genetic diversity in themtDNA control region and population structure in the pink shrimpFarfantepenaeus duorarum. Journal of Crustacean Biology 24: 101-109.

Morgan, E. M. J., B. S. Green, N. P. Murphy, and J. M. Strungnell.2013. Investigation of the genetic structure between deep and shallowpopulations of the southern rock lobster, Jasus edwardsii in Tasmania,South Australia. PLoS One 8: e77978.

Mullaney, T. J., and I. M. Suthers. 2013. Entrainment and retention of acoastal larval fish assemblage by a short-lived submescoscale, frontaleddy of the East Australian Current. Limnology and Oceanography 58:1549-1556.

Muths, D., E. Tessier, G. Gouws, M. Graig, M. Mwale, J. Mwaluma,A. Mwandya, and J. Bourjea. 2011. Restricted dispersal of the reef fishMyripristis berndti at the scale of the SW Indian Ocean. Marine EcologyProgress Series 443: 167-180.

Nylander, J. A. A. 2004. MrModeltest, version 2.2. Program distributed bythe author. Evolutionary Biology Centre, Uppsala University, Uppsala.

Ovenden, J. R., D. J. Brasher, and R. W. G. White. 1992. MitochondrialDNA analyses of the red rock lobster Jasus edwardsii support anapparent absence of population subdivision throughout Australasia.Marine Biology 112: 319-326.

, J. D. Booth, and A. J. Smolenski. 1997. Mitochondrial DNAphylogeny of red and green rock lobsters (genus Jasus). Marine andFreshwater Research 48: 1131-1136.

Palero, F., P. Abelló, E. Macpherson, M. Gristina, and M. Pascual. 2008.Phylogeography of the European spiny lobster (Palinurus elephas):influence of current oceanographical features and historical processes.Molecular Phylogenetics and Evolution 48: 708-717.

Panova, M., A. M. H. Blakeslee, W. A. Miller, T. Mäkinen, G. M. Ruiz,K. Johannesson, and C. André. 2011. Glacial history of the NorthAtlantic marine snail, Littorina saxatilis, inferred from distribution ofmitochondrial DNA lineages. PloS One 6: e17511.

Peakall, R., and P. E. Smouse. 2006. GENALEX 6: genetic analysis inExcel. Population genetic software for teaching and research. MolecularEcology Notes 6: 288-295.

Phillips, B. F., J. S. Cobb, A. Jeffs, and P. McWilliam. 2006. Larval andpostlarval ecology, pp. 231-262. In, B. F. Phillips (ed.), Lobsters: Bi-ology, Management, Aquaculture, and Fisheries. Blackwell Publishing,Oxford.

Pollock, D. E. 1992. Palaeoceanography and speciation in the spiny lobstergenus Panulirus in the Indo-Pacific. Bulletin of Marine Science 51: 133-146.

Rogers, A. R., and H. Harpending. 1992. Population growth makes wavesin the distribution of pairwise genetic differences. Molecular Biology andEvolution 9: 552-569.

Ronquist, F., and J. P. Huelsenbeck. 2003. MRBAYES 3: Bayesianphylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.

Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. G. Higuchi, G. T.Horn, K. B. Mullis, and H. A. Henlich. 1988. Primer-directed enzymaticamplification of DNA with a thermostable DNA polymerase. Science239: 487-491.

Silva, I. C., N. Mesquita, and J. Paula. 2010. Lack of population structure inthe fiddler crab Uca annulipes along an East African latitudinal gradient:genetic and morphometric evidence. Marine Biology 157: 1113-1126.

Steyn, E., and M. H. Schleyer. 2011. Movement patterns of the East Coastrock lobster Panulirus homarus rubellus on the coast of KwaZulu-Natal,South Africa. New Zealand Journal of Marine and Freshwater Research45: 85-101.

Strathmann, R. R., T. P. Hughes, A. M. Kuris, K. C. Lindeman, S. G.Morgan, J. M. Pandolfi, and R. R. Warner. 2002. Evolution of local

recruitment and its consequences for marine populations. Bulletin ofMarine Science 70: 377-396.

Tajima, F. 1989. Statistical method for testing the neutral mutation hypoth-esis by DNA polymorphism. Genetics 123: 585-595.

. 1993. Simple methods for testing the molecular evolutionary clockhypothesis. Genetics 135: 599-607.

Tavare, S. 1986. Some probabilistic and statistical problems in the analysisof DNA sequences, pp. 57-86. In, R. M. Miura (ed.), Some MathematicalQuestions in Biology – DNA Sequence Analysis. American Mathemati-cal Society, Providence, RI.

Teske, P. R., S. von der Heyden, C. D. McQuaid, and N. P. Barker. 2011.A review of marine phylogeography in southern Africa. South AfricanJournal of Science 107: 43-53.

Thomas, L., and J. J. Bell. 2013. Testing the consistency of connectivitypatterns for a widely dispersing marine species. Heredity 111: 345-354.

Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Clustal-W-improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position-specific gap penalties and weightmatrix choice. Nucleic Acids Research 22: 4673-4680.

Thorpe, J. P., A. M. Sole-Cava, and P. C. Watts. 2000. Exploited marineinvertebrates: genetics and fisheries. Hydrobiologia 420: 165-184.

Tolley, K. A., J. C. Groeneveld, K. Gopal, and C. A. Matthee. 2005.Mitochondrial DNA panmixia in spiny lobster Palinurus gilchristisuggests a population expansion. Marine Ecology Progress Series 297:225-231.

Toonen, R. J., and J. R. Pawlik. 2001. Foundations of gregariousness:a dispersal polymorphism among the planktonic larvae of a marineinvertebrate. Evolution 55: 2439-2454.

Tsang, L. M., Y. Achituv, K. H. Chu, and B. K. K. Chan. 2012.Zoogeography of intertidal communities in the West Indian Ocean asdetermined by ocean circulation systems: patterns from the Tetraclitabarnacles. PLoS One 7: e45120.

Volger, C., J. Benzie, P. H. Barber, M. V. Erdmann, Ambariyanto,C. Sheppard, K. Tenggardjaja, K. Gérard, and G. Wörheide. 2012.Phylogeography of the crown-of-thorns starfish in the Indian Ocean.PLos One 7: e43499.

von der Heyden, S., J. C. Groeneveld, and C. A. Matthee. 2007. Longcurrent to nowhere? – Genetic connectivity of Jasus tristani populationsin the southern Atlantic Ocean. African Journal of Marine Science 29:491-497.

, K. Prochazka, C. Rauci, and K. Bowie. 2008. Significant popula-tion structure and asymmetric gene flow patterns amidst expanding popu-lations of Clinus cottoides (Perciformes, Clinidae): application of molec-ular data to conservation panning in South Africa. Molecular Ecology 17:4812-4826.

Warner, R. R., and R. K. Cowen. 2002. Local retention of production inmarine populations: evidence, mechanisms and consequences. Bulletinof Marine Science 70: 245-249.

Warren, B. H., D. Strasberg, J. H. Bruggemann, R. P. Prys-Jones, andC. Thébaud. 2010. Why does the biota of the Madagascar region havesuch a strong Asiatic flavour? Cladistics 25: 526-538.

Xia, X., and P. Lemey. 2009. Assessing substitution saturation withDAMBE, pp. 615-630. In, P. Lemey, M. Salemi, and A. M. Vandamme(eds.), The Phylogenetic Handbook: A Practical Approach to DNAand Protein Phylogeny. 2nd Edition. Cambridge University Press, Cam-bridge.

Yednock, B. K., and J. E. Neigel. 2011. Rethinking the mechanisms thatshape marine decapod population structure, pp. 57-74. In, C. Held,S. Koenemann, and C. D. Schubart (eds.), Phylogeography and Popu-lation Genetics in Crustacean. CRC Press, Boca Raton, FL.

Yoder, A. D., and M. D. Nowak. 2006. Has vicariance or dispersal beenthe predominant biogeographic force in Madagascar? Only time will tell.Annual Review of Ecology, Evolution, and Systematics 37: 405-431.

Yokoyama, Y., K. Lambeck, P. D. Deckker, P. Johnston, and L. K. Fifield.2000. Timing of the last glacial maximum from observed sea levelminima. Nature 406: 713-716.

Zhang, D., and G. M. Hewitt. 1996. Nuclear integrations: challenges formitochondrial DNA markers. Trends in Ecology and Evolution 11: 247-251.

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