Journal of Biogeography (J. Biogeogr.) (2016) 43, 70–84

ORIGINAL The role of fire and a long-lived soil ARTICLE seed bank in maintaining persistence, genetic diversity and connectivity in a fire-prone landscape Donna Bradbury*, Sarah-Louise Tapper, David Coates, Shelley McArthur, Margaret Hankinson and Margaret Byrne

Science and Conservation Division, ABSTRACT Department of Parks and Wildlife, Bentley Aim Terrestrial ecology and evolution is significantly influenced by the Delivery Centre, Locked Bag 104, Kensington, phenomenon of fire, but studies of its potential impact on intraspecific genetic WA 6983, Australia variation and phylogeography are rare. This understanding will be important for predicting the biogeographical consequences of changing fire regimes under global climate change. Here, we asked whether changing historical fire regimes, together with climatic and geological history, have influenced phylogeographi- cal patterns in a fire-ephemeral vine. We also asked whether demographic stochasticity associated with a fire-ephemeral life history results in nuclear genetic drift as expected from spatio-temporal patchiness, or if this effect is buffered by the connectivity and diversity afforded by a persistent soil seed bank. Location The fire-prone, mediterranean-type climate region of south-western Australia. Methods We used Bayesian phylogeny reconstruction and statistical tests of demographic expansion based on variation at three non-coding chloroplast sequence regions (atpF, ndhF–rpl32, psbD–trnT) to reconstruct phylogeographi- cal history. Nuclear diversity and population structure at 11 microsatellite loci were investigated for evidence of genetic drift. Results Evidence for prolonged persistence and a lack of vicariance within the species range was found, together with strong evidence of historical demo- graphic expansion. Contrary to expectations, there was little evidence of nuclear genetic drift despite strong, above-ground spatio-temporal population patchiness. Main conclusions Our findings suggest that a late Pleistocene increase in fire frequency may have led to demographic expansion in this fire-ephemeral spe- cies; alternatively, the expansion signal may be an inherent feature of fire ephemerals with a persistent soil seed bank. Prolonged climatic stability has likely fostered persistence within the species range in contrast to contraction and vicariance. The notable lack of genetic drift implies a role for ample pollen dispersal and a long-lived soil seed bank in the maintenance of diversity and connectivity in an otherwise stochastic, fire-driven system. *Correspondence: Donna Bradbury, Science and Conservation Division, Department of Keywords Parks and Wildlife, Locked Bag 104, Bentley biodiversity hotpot, expansion, fire regime, coccinea, legume, Delivery Centre, Kensington, WA 6983, mediterranean-type climate, obligate seeder, phylogeography, soil seed bank, Australia. E-mail: [email protected] south-western Australia

70 http://wileyonlinelibrary.com/journal/jbi ª 2015 John Wiley & Sons Ltd doi:10.1111/jbi.12601 Phylogeographical structure of a fire-ephemeral vine

(Hopper & Gioia, 2004; Cowling et al., 2014), and the mesic INTRODUCTION biome is considered to be the ancestral biome of the conti- Plant diversification, adaptation and biogeography are known nent (Byrne et al., 2011). Given that such climatic stability to be significantly influenced by fire in strongly fire-prone in MCEs has contributed to the maintenance of exceptional regions of the globe (Pausas & Schwilk, 2012). While several diversity at the interspecific level (Cowling et al., 2014), spe- studies have investigated the effect of fire on speciation rates cies widespread within the High Rainfall Province are or the macro-evolution of fire-responsive plant traits (e.g. expected to exhibit high intraspecific diversity, together with He et al., 2011), studies of intraspecific genetic diversification long-term persistence characterized by isolation-by-distance in fire-responsive species are surprisingly rare, especially in (IBD) and a lack of strong regional genetic divergence or highly biodiverse regions of the Southern Hemisphere (Lexer structure. These patterns have largely been observed in two et al., 2013). A lack of such studies is particularly notable for forest tree species (Wheeler et al., 2003; Nevill et al., 2014) fire ephemerals, a life history type that is common in and in a herbaceous forest understorey species (Coates et al., mediterranean-climate ecosystems (MCEs) and defined here 2003). However, studies of widespread species characterized as short-lived, obligate-seeding species, where germination by a fire-driven distribution in this region are rare. and establishment from a soil seed bank is intrinsically It can be predicted that a fire-ephemeral life history may linked with fire. drive alternative phylogeographical and population genetic In Australia, fire has been a regular and ancient evolutionary patterns than those otherwise expected for co-occurring, less force within the landscape over long geological time-scales fire dependent perennial species. Recent reviews have (Crisp & Cook, 2013), including in the global biodiversity compared the obligate seeding (equivalent to fire ephemeral) hotspot of the Southwest Australian Floristic Region versus perennial, resprouting life history in fire-prone (SWAFR) (Hopper & Gioia, 2004). Here, charcoal evidence environments in a macroevolutionary context (Pausas & of regular fire is clearly apparent from at least the Pliocene Keeley, 2014), but little research has investigated how a fire- (Dodson et al., 2005) and the region contains many lineages ephemeral life history will shape intraspecific variation (e.g. of fire-responsive taxa (Crisp et al., 2011; He et al., 2011), Segarra-Moragues & Ojeda, 2010), making predictions including those with both canopy- and soil-stored seed difficult. It can be expected that strong fire-driven patchiness banks. Yet, given the paucity of genetic studies on fire- of ephemeral populations, in both space and time, is likely ephemeral species, it is difficult to predict the effects of such to result in genetic drift characterized by reduced nuclear a life history on patterns of genetic diversity. diversity within, and increased differentiation among, popu- Climate strongly influences fire regimes and therefore, fire lations relative to perennial species without such spatio- regimes have changed through time (Bowman et al., 2012). temporal population patchiness. These patterns are often For example, in south-eastern Australia, climatically dry peri- observed in species with fragmented distributions (Honnay ods in the Pleistocene are associated with increasing charcoal & Jacquemyn, 2007) and have been reported previously in a deposits, indicating increased fire (Kershaw et al., 2002), and species with a patchy, fire-driven distribution (Evans et al., there is evidence of more recent use of fire for landscape 2000). However, the presence of a stable, long-lived soil seed burning by Aboriginal people in the late Pleistocene (Turney bank may buffer populations from the demographic stochas- et al., 2001). Two Quaternary deposits from the mesic ticity experienced through repeated fire events (Honnay SWAFR reveal a significant increase in late Pleistocene and et al., 2008). In addition, strong fluctuations in population Late Glacial Maximum (LGM) charcoal representing a size of ephemeral species may leave genomic signatures that marked increase in fire (whether this represents an increase deviate from those expected in an otherwise historically in fire size, intensity or frequency is not known) (Pickett, stable environment. Given evidence for increased fire fre- 1997; Prideaux et al., 2010), and it is unclear whether this quency in the mesic SWAFR in the late Pleistocene (Pickett, increase was due to the Aboriginal use of fire, or increasing 1997; Prideaux et al., 2010), it is possible that fire-ephemeral aridity associated with the LGM, or both. The role of Abo- species in the region will show evidence of population riginal landscape burning in eliciting significant ecological growth and demographic expansions at this time, particularly change in Australia remains controversial (Bowman et al., for species with short juvenile periods and a persistent seed 2012), although recent genetic studies have concluded that bank. This would be in strong contrast to other studies in climate probably had a more dramatic impact on fire-prone the region that have noted demographic contraction events vegetation (Sakaguchi et al., 2013). due to increased aridity associated with glacial cycles (Byrne, Phylogeographical patterns of plant genetic diversity and 2008). structure in SWAFR are expected to be influenced by the Here, we treat the fire-ephemeral vine Kennedia coccinea long-term climatic and geological history of the region, as subsp. coccinea (Curtis) Vent. () as a model to test well as by the related effects of fire history. Similar to other predictions of how a fire-ephemeral life history may impact MCEs around the world, the mesic, forested High Rainfall genetic patterns in a fire-prone yet climatically stable envi- Province (sensu Hopper & Gioia, 2004) in the extreme ronment, using a phylogeographical and population genetic south-west of the SWAFR has experienced prolonged land- approach. The species is widespread in the understorey of scape and climatic stability relative to surrounding regions fire-prone jarrah forest in the High Rainfall Province of the

Journal of Biogeography 43, 70–84 71 ª 2015 John Wiley & Sons Ltd D. Bradbury et al.

SWAFR, and populations rapidly establish from a large, (2012) and PCR cycling was conducted as described by Shaw long-lived soil seed bank that is stimulated to germinate fol- et al. (2007), except that magnesium chloride concentration lowing fire, which kills adult . Above-ground plants are was 2.5 mm, and initial annealing temperature was 52 °C for short-lived (maximum 6 years) and become locally absent ndhF–rpl32 and psbD–trnT. PCR products were purified using without further fire or significant soil disturbance, leading to Agencourt AMPureXP paramagnetic beads (Beckman Coul- strong fire-driven spatial and temporal patchiness. We pre- ter, USA), sequenced by Macrogen Inc. (Seoul, South Korea) dict there will be phylogeographical evidence of widespread and visualized in ABI Sequence Scanner 1.0 (Applied persistence of the species throughout the region, consistent Biosystems 2005) to assess quality. Sequences were checked with long-term stability, but that features of a fire-ephemeral for miscalls in Sequencher 5.0 (Genecodes Corp., USA), life history, such as population patchiness, will result in evi- aligned using ClustalW 1.4 (Thompson et al. 1994) and dence of population size fluctuation and contemporary manually where necessary, and trimmed to equal lengths genetic drift. However, a persistent soil seed bank may before all three regions were concatenated in mega5.2.2 reduce the impact of genetic drift by maintaining population (Tamura et al., 2011). Insertion–deletions (indels) were man- genetic diversity and connectivity. ually coded as binary characters at the end of the sequence data set, although homopolymer-length differences were ignored due to a high likelihood of read errors. MATERIALS AND METHODS A total of 439 individuals from 22 populations were geno- typed using 11 nuclear genomic microsatellite markers, Study system according to Bradbury et al. (2014). Kennedia belongs to the globally distributed, economically important tribe of the Fabaceae family. Three Data analyses subspecies distinguished by floral characteristics have been recently described (Lally, 2010); for simplicity, we focus this Chloroplast haplotype diversity study on K. coccinea subsp. coccinea, the common, wide- spread subspecies that occurs throughout the forest, herein Haplotype and nucleotide diversity (HD, p) were calculated referred to simply as K. coccinea. in Arlequin 3.5.1.2 (Excoffier & Lischer, 2010). Ordered

Populations of K. coccinea have a widespread but patchy and unordered within-population diversity (vS,hS), total distribution within the understorey of fire-prone jarrah for- diversity (vT, hT) and population differentiation coefficients ests that are continuously distributed over 400 km. Mass- (NST, GST) were calculated using permut 2.0 (Pons & Petit, flowering of K. coccinea occurs in spring and mature fruits 1996). Unordered analyses consider only allele frequencies in develop through summer. Flowers are orange-red-pink and estimating diversity and differentiation (all haplotypes are of the papilionoid ‘pea’ morphology, where reproductive considered equally divergent), but ordered analyses also take structures are enclosed within the keel and exposed upon into account distances between alleles (haplotype sequence floral handling. In such systems pollination is effected only similarity). by animal vectors, mainly bees. The hard-coated seeds are dispersed close to the maternal plant via ejection from spi- Tests of phylogeographical structure rally dehiscing fruits, although the presence of an elaiosome suggests secondary dispersal by ants. Seeds are likely to be We used permut to test whether NST was significantly very long-lived in the seed bank, similar to other hard-coated greater than GST, indicating whether haplotypes within popu- leguminous species from seasonally dry mediterranean-type lations are more likely to be closely related than haplotypes environments (Daws et al., 2007; Gibson et al., 2011). among populations. The result can be sensitive to sampling artefacts, thus our high number of populations and homoge- nous sample size within populations will make an interpreta- Sampling and genotyping tion of phylogeographical structure more robust (Pons & Leaf material was collected from 439 individuals from 22 Petit, 1996). A spatial analysis of molecular variance populations, spanning 340 km (Table 1). Nuclear genomic (SAMOVA) was conducted using samova 1.0 (Dupanloup DNA was extracted from freeze-dried material according to et al., 2002) to characterize phylogeographical structure, Doyle & Doyle (1987) with addition of 1% w/v which defines K groups of populations that are geographi- polyvinylpyrrolidone (MW 40000) to the extraction buffer. cally homogeneous and maximally differentiated, based on a

Non-coding chloroplast DNA (cpDNA) regions known to procedure that maximizes FCT (proportion of total genetic detect phylogeographical structure (Byrne & Hankinson, variance due to differences between groups). K was modelled 2012) were trialled, and two intergenic spacer regions (psbD– from 2 to 20 with 100 simulations each. Optimal K was trnT; ndhF–rpl32) (Shaw et al., 2007) and one D4-loop intron identified when FCT plateaued at its highest value. (atpF) (Watts et al., 2008) were selected for further analysis Relationships among chloroplast haplotypes were deter- and sequenced in 176 individuals from 22 populations. mined by constructing a median-joining maximum parsimony Sequences were amplified according to Byrne & Hankinson (MJMP) network in Network 4.6.1.2 (Bandelt et al., 1999).

72 Journal of Biogeography 43, 70–84 ª 2015 John Wiley & Sons Ltd Phylogeographical structure of a fire-ephemeral vine

Table 1 Characteristics of 22 Kennedia coccinea subsp. coccinea populations from south-western Australia used for genotyping.

Population Code n Location Elevation (m) Rainfall isohyet (mm) Geomorphological zone

Toodyay Too 20, 8 31°38022.0″ S 268 700–800 EDR 116°23016.8″ E Bailup Bail 20, 8 31°44017.9″S 300 800–900 EDR 116°17041.2″ E Chidlow Chid 20, 8 31°52030.8″ S 301 600–700 EDR 116°23041.9″ E Maidavale Maid 20, 8 31°57037.3″ S 94 800–900 P 116°01022.3″ E Karragullen Kar 20, 8 32°06040.2″ S 288 1200–1300 WDR 116°08027.9″ E Gleneagle Glen 20, 8 32°17012.0″ S 321 1100–1200 WDR 116°11027.2″ E Jarrahdale Jar 20, 8 32°19054.0″ S 302 1200 WDR 116°05012.0″ E Kingsbury King 20, 8 32°24047.8″ S 249 1000–1100 WDR 116°00032.0″ E Pinjarra Pinj 20, 8 32°42022.3″ S 192 1300 WDR 115°59023.9″ E Yarloop Yar 20, 8 32°56026.0″ S 32 1000–1100 P 115°54032.6″ E Harris Har 20, 8 33°20036.6″ S 208 900–1000 WDR 116°08043.2″ E Collie Coll 20, 8 33°20042.6″ S 215 900–1000 WDR 116°10056.1″ E Kirup Kir 20, 8 33°43028.3″ S 239 1000 WDR 115°54007.8″ E Payne Pay 20, 8 33°45003.3″ S 51 1000–1100 P 115°13028.0″ E McAtee Mcat 20, 8 33°57051.6″ S 135 900–1000 DS 115°31005.1″ E Brockman Broc 20, 8 34°09035.4″ S 48 1000–1100 DS 115°17021.6″ E Perup Per 20, 8 34°11009.3″ S 276 600–700 WDS 116°37056.4″ E Graphite Grap 20, 8 34°12052.0″ S 236 1200–1300 WDS 115°57030.8″ E Manjimup Manj 19, 8 34°13022.3″ S 274 1000–1100 WDS 116°07005.5″ E Wheatley Whe 20, 8 34°20030.9″ S 199 900–1000 WDS 116°16019.7″ E Northcliffe Nor 20, 8 34°38005.9″ S 103 1400 WDS 116°07057.6″ E Bevan Bev 20, 8 34°39012.2″ S 199 900–1000 WDS 116°58026.0″ E n – number of samples for nuclear and chloroplast analyses respectively; Location – site latitude and longitude; Elevation – site elevation in metres; Rainfall isohyet – site annual rainfall based on nearest isohyet line(s); Geomorphological zone – a soil geomorphological classification unit that reflects broad erosion/deposition patterns and landscape maturity: EDR – Eastern Darling Range Zone; WDR – Western Darling Range Zone; P – Pinjarra Zone; DS – Donnybrook Sunkland Zone; WDS – Warren-Denmark Southland Zone (Schoknecht et al., 2004).

Bayesian phylogeny reconstruction of haplotypes was con- Two haplotypes of Kennedia coccinea subsp. esotera were ducted in MrBayes 3.1.2 (Ronquist et al., 2012) using four included in the tree as outgroups. Markov chain Monte Carlo (MCMC) chains for 1,000,000 generations. Bayesian analysis adopted the SYM substitution Tests of expansion model with gamma rate variation, which was determined as the best fit by jModelTest 0.1.1 (Guindon & Gascuel, 2003). To assess likelihood of departures from neutrality and popu-

Binary indel coding was separately partitioned and analysed lation growth, Tajima’s (1989) D and Fu’s (1997) FS were under a simple JC model. A 50% majority consensus tree was calculated using Arlequin, and Ramos-Onsins & Rozas’s constructed with the first 25% of trees discarded as burnin. (2002) R2 was calculated using DnaSP 5.1.1 (Librado &

Journal of Biogeography 43, 70–84 73 ª 2015 John Wiley & Sons Ltd D. Bradbury et al.

Rozas, 2009). The D and R2 statistics are based on the presence of spatial autocorrelation (IBD). We ran models frequency spectrum of polymorphic nucleotide sites, where with (BYM model) and without admixture, using 50,000 sudden population growth is expected to generate an excess of sweeps after a burn-in of 10,000, modelling Kmax from 2 to rare, weakly diverged mutations (Ramos-Onsins & Rozas, 23, including 100 runs of each Kmax, applying a spatial inter- 2002). The FS statistic is based on haplotype distribution. All action factor of 0.6. The most likely K was determined based values are expected to be low or negative when there is an on the explanation in Appendix S1 in the Supporting excess of rare mutations. Positive selection can also produce Information. The Greedy algorithm within clumpp 1.1.2 significant values of these statistics, therefore, we calculated (Jakobsson & Rosenberg, 2007) and distruct 1.1 (Rosen- them for all three chloroplast regions independently. Low or berg, 2004), were used to respectively obtain, and plot, the significant values for multiple regions support expansion optimal alignment of the q-matrix of individual cluster rather than selection, given that demographic effects influence membership proportions. the whole genome similarly, but selection is locus-specific. Mismatch distributions were also plotted using Arlequin. RESULTS Deviation of observed values from those expected under a sce- nario of sudden spatial or demographic expansion was Chloroplast haplotype diversity assessed using both the raggedness index (HRAG) (Harpending, 1994) and the sum of squared deviations between observed The atpF, ndhF–rpl32 and psbD–trnT regions were 648 bp, and expected distribution (SSD), using Arlequin. Given that 842 bp and 1536 bp in length respectively. Across all 3026 coded indels may inflate estimates of recent mutations, they bases of sequence in 176 K. coccinea individuals there were were excluded from all tests of expansion. 264 polymorphic sites, including 35 indels. A total of 46 unique haplotypes were identified, 39 of which (85%) were specific to single populations (Table 2). Accordingly, mea- Nuclear diversity sures of total haplotype diversity were extremely high Nuclear genetic diversity parameters were calculated using (Table 3), while diversity within populations was comparably genalex 6.4 (Peakall & Smouse, 2006). Allelic richness per lower. population was calculated using fstat 2.9.3.2 (Goudet, 2001). Global tests of heterozygote deficiency, and tests of Tests of phylogeographical structure linkage disequilibrium (LD) among pairs of loci, were per- formed with genepop 4.2 (Rousset, 2008), applying Bonfer- Both ordered and unordered population differentiation roni correction. Frequency of null alleles was estimated using indices were significantly greater than zero, strongly indica- the EM algorithm with FreeNA (Chapuis & Estoup, 2007), tive of significant population structure (Table 3). Further, as was the level of among-population differentiation (FST) NST was significantly greater than GST (P = 0.000) (Table 3), per locus. To determine whether null alleles had a significant indicating that strongly related haplotypes were more often effect on estimates of differentiation, FreeNA was used to found together within populations than less related haplo- calculate per-locus FST both with and without the ENA types, reflecting significant phylogeographical structure at the correction method, using 1000 bootstraps to generate 95% population level. It is possible that variable mutation rates confidence intervals. and/or excess of rare haplotypes in the data set (see ‘Tests of

expansion’ below) has biased the value of GST downwards, possibly influencing the relative significance of the test (Pons Nuclear population structure and connectivity & Petit, 1996). However, an explanation of significant phylo-

Pairwise FST was calculated for each population pair using geographical structure is most likely when the results are fstat. The relative distribution of genetic variation within considered together with the SAMOVA result, the relatedness and among populations was assessed by an analysis of of haplotypes within populations as exemplified in the net- molecular variance (AMOVA) using genalex, with 1000 work (Fig. 1) (see below) and the lack of haplotype sharing permutations. IBD was tested by a correlation between geo- among populations (Fig. 2). graphical distance and pairwise population genetic differenti- SAMOVA could not identify an appropriate value of K ation (FST/1 FST) using a Mantel procedure in genalex. that maximized FCT, which continued to increase, without Patterns of population genetic structure were visualized clearly plateauing, to a maximum allowable level of K = 20, using Nei’s (1978) genetic distance matrix to construct an where 74% of the total chloroplast haplotype variation was unweighted pair group method with arithmetic mean explained by differences among groups (essentially, among (UPGMA) tree using gda 1.1 (Lewis & Zaykin, 2001). To populations). characterize spatial structure based on hierarchical Bayesian The haplotype network was complex, with several exam- modelling, tess 2.3.1 (Chen et al., 2007) was used to deter- ples of closed loops indicative of homoplasy, making some mine the most likely number (Kmax) of genetically homoge- relationships ambiguous. The network formed an approxi- nous clusters, and to assign a proportionate membership (q) mate star-like structure which placed the most common and of each individual to each cluster, while accounting for geographically widespread haplotype, H05, in a central and

74 Journal of Biogeography 43, 70–84 ª 2015 John Wiley & Sons Ltd Phylogeographical structure of a fire-ephemeral vine

Table 2 Chloroplast haplotypes defined from sequences of two intergenic spacer regions (ndhF–rpl32, psbD–trnT) and one D4-loop intron (atpF)inKennedia coccinea subsp. coccinea from eight individuals from 22 populations in south-western Australia. Two haplotypes of Kennedia coccinea subsp. esotera are provided as outgroups.

GenBank accession number

Haplotype Location (number of individuals) Frequency (%) atpF ndhF–rpl32 psbD–trnT

H01 Bailup (7) 3.98 KM879931 KM879973 KM879943 H02 Bailup (1) 0.57 KM879932 KM879973 KM879943 H03 Bevan (8) 4.55 KM879933 KM879974 KM879944 H04 Brockman (4) 2.27 KM879933 KM879975 KM879945 H05 Perup (7), Kirup (6), Brockman (3), 11.36 KM879933 KM879976 KM879945 Yarloop (3), Northcliffe (1) H06 Brockman (1) 0.57 KM879933 KM879977 KM879945 H07 Chidlow (2) 1.14 KM879933 KM879978 KM879946 H08 Chidlow (1) 0.57 KM879934 KM879979 KM879947 H09 Chidlow (2) 1.14 KM879933 KM879980 KM879946 H10 Chidlow (1) 0.57 KM879933 KM879980 KM879948 H11 Chidlow (1) 0.57 KM879934 KM879979 KM879949 H12 Chidlow (1) 0.57 KM879934 KM879979 KM879950 H13 Collie (6), Harris (1) 3.98 KM879935 KM879981 KM879951 H14 Collie (2) 1.14 KM879933 KM879981 KM879951 H15 Gleneagle (4) 2.27 KM879931 KM879982 KM879952 H16 Karagullen (4), Gleneagle (1) 2.84 KM879936 KM879983 KM879953 H17 Gleneagle (1) 0.57 KM879931 KM879984 KM879952 H18 Gleneagle (1) 0.57 KM879937 KM879985 KM879954 H19 Gleneagle (1) 0.57 KM879938 KM879986 KM879955 H20 Graphite (7), Wheatley (7), Northcliffe (5) 10.80 KM879931 KM879987 KM879956 H21 Graphite (1) 0.57 KM879931 KM879988 KM879956 H22 Harris (4) 2.27 KM879931 KM879989 KM879944 H23 Harris (3), Perup (1) 2.27 KM879931 KM879990 KM879944 H24 Jarrahdale (4) 2.27 KM879939 KM879991 KM879957 H25 Jarrahdale (3) 1.70 KM879940 KM879991 KM879957 H26 Jarrahdale (1) 0.57 KM879941 KM879992 KM879958 H27 Karagullen (2) 1.14 KM879933 KM879976 KM879959 H28 Karagullen (1) 0.57 KM879935 KM879993 KM879960 H29 Karagullen (1) 0.57 KM879935 KM879983 KM879961 H30 Kingsbury (4) 2.27 KM879931 KM879976 KM879962 H31 Kingsbury (1) 0.57 KM879931 KM879994 KM879963 H32 Kingsbury (1) 0.57 KM879931 KM879980 KM879964 H33 Kingsbury (2) 1.14 KM879931 KM879995 KM879962 H34 McAtee (8), Kirup (2) 5.68 KM879936 KM879994 KM879967 H35 Maidavale (7) 3.98 KM879931 KM879990 KM879965 H36 Maidavale (1) 0.57 KM879936 KM879996 KM879966 H37 Manjimup (8), Northcliffe (2), Wheatley (1) 6.25 KM879931 KM879997 KM879944 H38 Payne (2) 1.14 KM879931 KM879998 KM879968 H39 Payne (3) 1.70 KM879931 KM879994 KM879968 H40 Payne (3) 1.70 KM879931 KM879999 KM879968 H41 Pinjarra (6) 3.41 KM879931 KM880000 KM879969 H42 Pinjarra (2) 1.14 KM879931 KM880001 KM879969 H43 Toodyay (7) 3.98 KM879942 KM880002 KM879970 H44 Toodyay (1) 0.57 KM879942 KM880003 KM879970 H45 Yarloop (4) 2.27 KM879931 KM880004 KM879971 H46 Yarloop (1) 0.57 KM879936 KM880005 KM879972 Outgroup H1 K. coccinea subsp. esotera KM880006 KM880007 KM880008 Outgroup H2 K. coccinea subsp. esotera KM880006 KM880007 KM880009

presumably ancestral position within the network, with 10 one other haplotype. An inferred intermediate, unsampled connections of both long and short branch lengths (Fig. 1). haplotype also had 10 connections, most of which led to The second-most common haplotype (H20) was restricted to haplotypes found in the north. Haplotypes in the south were the south-east (Fig. 2) and was inferred to give rise to just more likely to be shared, and were more closely related to

Journal of Biogeography 43, 70–84 75 ª 2015 John Wiley & Sons Ltd D. Bradbury et al. the ancestral H05 (minimum 3.8 0.5 mutations) than haplotypes were restricted to this group. Therefore, there was haplotypes found in the north (minimum 5.9 0.4 muta- not a strong geographical pattern to relationships evident in tions), although there were exceptions to this general pattern the network overall, nor were there clear divergences of (e.g. H27 in Karagullen). One large group of haplotypes was regional haplotype lineages. restricted to six northern populations, but not all northern The phylogenetic tree had a brush-like structure with multiple polytomies, and a lack of clear regional lineage Table 3 Diversity and differentiation indices for chloroplast structure (Fig. 3). Clades were rarely concordant with geog- haplotype data from psbD–trnT, ndhF–rpl32 and atpF sequences raphy, with the exception that most (5 of 7) of the basal of Kennedia coccinea subsp. coccinea in eight samples from 22 clades were found in southern populations, and one large populations in south-western Australia. clade was restricted to six northern populations (as in the Test/Measure Statistic Value network). However, haplotypes from all northern popula- tions were not restricted to the same clade (e.g. Maidavale, Haplotype diversity HD 0.957 (0.006) Karragullen). Nucleotide diversity p 0.002 (0.001)

Within-population diversity hS 0.458 (0.061) (unordered) Tests of expansion Within-population diversity vS 0.264 (0.055) (ordered) Values of D, FS and R2 were all low and highly significant Total diversity (unordered) hT 0.978 (0.014) (Table 4), indicating a significant excess of rare, weakly Total diversity (ordered) vT 0.986 (0.117) diverged haplotypes, consistent with the predicted effects of Population differentiation G 0.532 (0.061) ST demographic expansion. Values of each statistic for each (unordered) independent chloroplast region were also low or negative, Population differentiation NST 0.733 (0.054) (ordered) favouring an explanation of expansion over locus-specific

Phylogeographical structure NST >GST P = 0.000 selection (see Appendix S2). The mismatch HRAG and SSD values also did not deviate from a model of sudden expan- Standard errors in parentheses. sion (Table 4). The values under a demographic versus

Figure 1 Phylogenetic network of 46 chloroplast haplotypes observed in Kennedia coccinea subsp. coccinea from south-western Australia. Haplotypes were identified from analysis of psbD–trnT, ndhF–rpl32 and atpF sequences in eight samples from 22 populations. The central haplotype (H05) is inferred as ancestral. Circle sizes are proportional to haplotype frequency. Small black dots represent a single nucleotide substitution or indel. White diamonds are inferred unsampled haplotypes. Population name codes correspond to those in Table 1. Haplotype numbers correspond to those in Table 2. Colours correspond to those in Figs 2 and 3.

76 Journal of Biogeography 43, 70–84 ª 2015 John Wiley & Sons Ltd Phylogeographical structure of a fire-ephemeral vine

Figure 2 Distribution of Kennedia coccinea subsp. coccinea chloroplast haplotypes, overlaid on a geographical map of sampling locations in south-western Australia. Haplotypes were identified from analysis of psbD–trnT, ndhF–rpl32 and atpF sequences in eight samples from 22 populations. Proportions in the pie charts represent the proportion of individuals in the sampled population that possess a given haplotype. Population name codes correspond to those in Table 1. Haplotype colours correspond to those in Figs 1 and 3. spatial expansion model were highly similar, thus one sce- south-east, and highest in the combined north-west and nario could not be rejected over the other. south-west (Fig. 4b).

Nuclear diversity Nuclear structure

All loci were highly variable, exhibiting between 7 and 37 AMOVA (FST = 0.05, P = 0.001) and pairwise FST values alleles per locus (see Appendix S3). Measures of corrected (range 0.000–0.134; mean = 0.06) revealed the presence of

FST per locus ranged from 0.030 to 0.204. Linkage disequi- significant, but low, nuclear structure throughout the species librium between up to 17 pairs of loci was detected in range. In contrast to the SAMOVA of chloroplast markers, nine of the 22 populations. All but two populations AMOVA attributed just 5% of the total nuclear variation to exhibited significant heterozygote deficiency and relatively differences among populations. Significant structure was high fixation indices (Table 5). This high level of fixation observed in the form of strong IBD, such that geographically may be explained in part by the presence of null alleles at proximate populations were more closely related than geo- relatively high frequencies for some loci (Appendix S3), graphically distant populations (Fig. 4c). The distance-based but the overall FST of the data set was not significantly UPGMA (Fig. 4b) analysis grouped the populations similarly different before (0.056) (95% CI: 0.040–0.082) or after into three broad groups: a far northern group (Bailup, Too- (0.054) (95% CI: 0.038–0.080) null allele correction. dyay), a south-eastern group (Graphite, Manjimup, Wheat- Nuclear diversity was distributed relatively randomly ley, Perup, Northcliffe, Bevan) and a central group (all other among populations throughout the range (Table 5), populations). Maidavale was further distinguished from the although on a regional scale, allelic richness tended to be central group by UPGMA and tess. Within the central lowest in the two far northern populations, higher in the group there was a potential further subdivision forming

Journal of Biogeography 43, 70–84 77 ª 2015 John Wiley & Sons Ltd D. Bradbury et al.

Figure 3 Bayesian haplotype phylogeny of 46 chloroplast haplotypes observed in Kennedia coccinea subsp. coccinea from south-western Australia. Haplotypes were identified from analysis of psbD–trnT, ndhF–rpl32 and atpF sequences in eight samples from 22 populations. Numbers at nodes indicate posterior probabilities. Two haplotypes of Kennedia coccinea subsp. esotera are included as outgroups. Broken branches indicate a reduced branch length for illustrative purposes. Colours correspond to those in Figs 1 and 2.

north-west and south-west components. The model-based persisted within a region with stable climate. Significant evi- tess analysis revealed that the optimal number of population dence of demographic expansion may be due to the presence clusters was Kmax = 5, comprising far north, north-west, of a persistent soil seed bank maintaining rare diversity or to south-west and south-east clusters, plus the Maidavale popu- the fire-ephemeral life history leading to rapid demographic lation (Fig. 4, Appendix S1). Similar to the distance-based population growth in response to fire. Patterns of localized methods, the far north and south-east clusters were more haplotype variation appeared to be associated with localized clearly differentiated than the north-western and south-west- geographical variation in fire frequency. Notably, a lack of ern clusters, which were more strongly admixed (Fig. 4), evidence for genetic drift highlighted the likely role of a large probably reflective of a continuous cline of IBD in the core and long-lived soil seed bank in maintaining diversity and distributional range. connectivity in an otherwise patchy, ephemeral system.

DISCUSSION Prolonged persistence

Fire and its interaction with a soil seed bank, together with Consistent with predictions for prolonged and widespread the climatic history of the High Rainfall Province of the persistence within a climatically stable region (e.g. Cowling SWAFR, appears to have had significant impacts on the phy- et al., 2014), K. coccinea exhibited high genetic diversity and logeographical structure of K. coccinea. We found evidence endemism (particularly of chloroplast markers), significant for prolonged persistence within the species’ range, including phylogeographical structure to the level of populations rather high genetic diversity (especially of the chloroplast genome) than regions, a lack of vicariance signals in the haplotype and a lack of evidence for vicariance or divergence of regio- network or phylogeny, strong nuclear IBD, and low nuclear nal lineages, consistent with predictions for a species that has among-population differentiation. This combination of

78 Journal of Biogeography 43, 70–84 ª 2015 John Wiley & Sons Ltd Phylogeographical structure of a fire-ephemeral vine

Table 4 Tests of expansion in analysis of psbD–trnT, ndhF– by the fire ephemeral life history, and possibly by the fire rpl32 and atpF chloroplast sequences of Kennedia coccinea subsp. history of the region. Signals of expansion are a novel find- coccinea in eight samples from 22 populations from south- ing in the context of prolonged and widespread species per- western Australia. sistence, given that expansion signals are often interpreted to Statistic Value reflect rapid historical range expansion events following con- traction to glacial refugia (Hewitt, 2000). However, we do Tajima’s Da 1.777 (P = 0.006) a not find evidence for significant contraction events or subse- Ramos-Onsin and Rozas’s R2 0.034 (P = 0.027) b quent range expansion in K. coccinea in the patterns of phy- Fu’s FS 16.130 (P = 0.000) c logeographical or population genetic diversity and structure. D-HRAG 0.017 (P = 0.786) D-SSD 0.001 (P = 0.789)c A strong expansion signal in a fire-ephemeral species can c S-HRAG 0.017 (P = 0.786) be explained by two scenarios. First, this result is consistent S-SSD 0.001 (P = 0.720)c with historical, rapid demographic population growth and increased generation turnover within the species’ range (not D-HRAG – demographic expansion model, mismatch raggedness index; D-SSD – demographic expansion model, mismatch sum of necessarily spatial range expansion), in response to an squared deviations; S-HRAG – spatial expansion model, mismatch increase in fire frequency, which is known to have occurred raggedness index; S-SSD – spatial expansion model, mismatch sum in the SWAFR region in the late Pleistocene (Pickett, 1997; of squared deviations. Prideaux et al., 2010). No studies of perennial plant species a D and R2 statistics are based on the frequency spectrum of poly- in the broader SWAFR region to date have identified such a morphic nucleotide sites, where sudden population growth is strong signal of expansion in the absence of signals of con- expected to generate an excess of rare, less highly diverged (recent) traction (Byrne, 2008; Nistelberger et al., 2014), suggesting mutations, resulting in negative and low values respectively. b that this signal may therefore be unique to fire ephemerals, The FS statistic is based on the haplotype distribution, and has a negative value when there is an excess of rare mutations. particularly those with short juvenile periods, although con- cThe models test deviation of observed values from an expected dis- firmation will require more research on species with similar tribution under population expansion. Non-significant P-values do life histories. not deviate from the null model and support a scenario of expansion. A second explanation is that an expansion signal may be an inherent feature of short-lived, fire-responsive species with a soil seed bank that has evolved under recurring fire, features is not expected to form in a taxon that has recently regardless of whether the local fire frequency historically expanded its geographical range, nor in response to signifi- increased. For example, soil seed banks are known to act as cant range contraction. Instead, the data suggest predomi- reservoirs of rare alleles (Honnay et al., 2008), which, when nantly in-situ evolution of genetic diversity from a common they repeatedly arise in above-ground populations following ancestor over time. mass post-fire establishment, may contribute to the excess of The presence of significant IBD and low differentiation rare but weakly diverged haplotypes that drive significant among nuclear genetic clusters also implies a relatively long- values of the D, FS and R2 statistics. The ongoing process of term stability to the overall patterns of gene flow and popu- highly localized, transient establishment of above-ground lation distribution – patterns that are largely consistent with populations, replicated throughout the species’ range after those seen in previous studies of widespread species in the regular local fire events, is therefore also likely to produce forested High Rainfall Province (Coates et al., 2003; Wheeler such a signal of demographic expansion in the absence of an et al., 2003). However, the location of the south-eastern increase in actual fire frequency, and in the absence of a spa- nuclear genetic cluster in K. coccinea was notable, given its tial range expansion or notable increase in total effective apparent demarcation by the Blackwood River system, south population size. Further research on other fire-ephemeral of which the broad geomorphological zone of the region also species worldwide, including in regions that have not experi- changes (Schoknecht et al., 2004). The Blackwood River has enced a marked increase in historical fire frequency, would been identified as a boundary between lineages within two shed further light on this hypothesis. This hypothesis would small animal species (Gouws et al., 2006; Edwards et al., also benefit from a direct comparison with perennial species 2008), although chloroplast lineage structure in jarrah did in fire-prone environments, which we expect will not show a not follow the same pattern (Wheeler & Byrne, 2006). The strong expansion signal due to the absence of regular, cyclical river may therefore act as a stronger barrier to gene flow for post-fire recruitment from a seed bank. understorey species and small animals than it does for large, dominant forest trees. Fire and haplotype diversity

Greater haplotype sharing, and lower haplotype diversity, in Fire and expansion the south-east of the range relative to the north may be related A scenario of sudden demographic expansion was evident in to less frequent fire. In a fire-ephemeral species with a large the significant excess of rare, weakly diverged chloroplast and long-lived soil seed bank, higher fire frequency results in haplotypes. Such a signal of expansion may have been driven more frequent population turnover and opportunity for novel

Journal of Biogeography 43, 70–84 79 ª 2015 John Wiley & Sons Ltd D. Bradbury et al.

Table 5 Nuclear genetic diversity characteristics of Kennedia coccinea subsp. coccinea populations from south-western Australia derived from analysis of 11 microsatellite markers in 20 individuals from 22 populations.

Population AT SAR HO HE FIS

Toodyay 60 0 4.7 0.60 (0.07) 0.64 (0.06) 0.044 (0.080) Bailup 65 3 5.1 0.53 (0.05) 0.63 (0.05) 0.143 (0.069)* Chidlow 95 6 7.0 0.53 (0.08) 0.72 (0.07) 0.223 (0.093)* Maidavale 70 1 5.2 0.56 (0.09) 0.62 (0.08) 0.088 (0.084) Karragullen 89 1 6.7 0.52 (0.09) 0.72 (0.06) 0.260 (0.096)* Gleneagle 91 0 6.7 0.57 (0.06) 0.71 (0.06) 0.165 (0.066)* Jarrahdale 99 4 7.1 0.55 (0.07) 0.74 (0.05) 0.232 (0.089)* Kingsbury 85 2 6.5 0.67 (0.06) 0.73 (0.05) 0.055 (0.092)* Pinjarra 101 3 7.2 0.61 (0.07) 0.75 (0.05) 0.182 (0.075)* Yarloop 84 0 6.3 0.55 (0.06) 0.71 (0.06) 0.209 (0.070)* Harris 101 2 7.1 0.52 (0.07) 0.70 (0.69) 0.239 (0.081)* Collie 90 2 6.5 0.46 (0.07) 0.67 (0.08) 0.303 (0.070)* Kirup 98 2 7.2 0.55 (0.09) 0.71 (0.08) 0.228 (0.075)* Payne 88 1 6.7 0.55 (0.08) 0.72 (0.06) 0.222 (0.090)* McAtee 98 0 7.0 0.55 (0.08) 0.71 (0.07) 0.199 (0.074)* Brockman 91 3 6.9 0.58 (0.08) 0.72 (0.07) 0.205 (0.076)* Perup 85 0 6.4 0.54 (0.08) 0.70 (0.05) 0.232 (0.092)* Graphite 88 2 6.4 0.57 (0.07) 0.70 (0.04) 0.180 (0.086)* Manjimup 87 3 6.8 0.59 (0.09) 0.75 (0.04) 0.219 (0.093)* Wheatley 85 1 6.3 0.60 (0.07) 0.72 (0.05) 0.152 (0.086)* Northcliffe 76 1 6.1 0.57 (0.07) 0.72 (0.04) 0.234 (0.077)* Bevan 95 1 7.3 0.52 (0.08) 0.76 (0.04) 0.304 (0.092)* Mean 87 (2) 1.7 (0.3) 6.5 (0.1) 0.55 (0.01) 0.71 (0.01) 0.196 (0.014)

AT – total number of alleles observed; S – number of private alleles; AR – Allelic richness of each population based on a minimum sample size of 10 individuals; HO – observed heterozygosity; HE – expected heterozygosity; FIS – fixation index. *Significant deviation from the Hardy–Weinberg equilibrium (heterozygote deficiency) (P < 0.01). and rare diversity from the seed bank to arise in above-ground differentiation were observed, features that are normally seen populations, relative to areas that have less frequent fire. It is in widespread species with continuous distributions. We possible that this pattern is driven by ecological factors other therefore propose that the continuity, and diversity, provided than fire frequency (e.g. effective population size), but indirect by a persistent soil seed bank allows K. coccinea to function evidence for lower fire frequency in the south-eastern region on some levels as a common, widespread species despite its supports our interpretation. Presently, tall wet forest domi- patchy, stochastic above-ground distribution, together with a nated by Eucalyptus diversicolor (which occurs in the vicinity role of ample effective pollen dispersal in maintaining low of the south-eastern cluster of populations) is less prone to fire differentiation. The level of chloroplast haplotype diversity than other eucalypt forest and woodland (the remainder of the was particularly striking and unexpected, and was surpris- K. coccinea range) (Murphy et al., 2013). In addition, there is ingly higher than that reported in a perennial species com- evidence that southern tall wet forests were less utilized by plex with a vast range across the SWAFR (Nistelberger et al., Aboriginal people, and therefore less frequently burnt, than 2014), further suggesting that a persistent soil seed bank may the northern jarrah forest and forest margins (Burrows et al., function to retain high levels of diversity. 1995; Hassell & Dodson, 2003). Recent work confirms the role of soil seed banks as reser- voirs, or even accumulators, of genetic diversity (Dolan et al., 2008; Roberts et al., 2014), and we argue that this may Seed bank promotes diversity and connectivity also be the case in K. coccinea, via the following mechanisms. The persistence (through time) and continuity (through Given the predicted longevity of hard-coated legume seeds, it space) of a large, long-lived soil seed bank appears to have is likely that only a proportion of the seed bank germinates buffered above-ground populations from predicted effects of after any given fire, depending on species and fire character- genetic drift, which would otherwise be expected for a taxon istics (Auld & Denham, 2006), enabling persistence of the with strong spatio-temporal patchy distribution. We expected seed bank even in the presence of short inter-fire intervals. to see some evidence of drift, such as low nuclear diversity, Relative to other jarrah forest species, K. coccinea can germi- high among-population differentiation or geographically ran- nate over a wide range of seed burial depths (Grant et al., dom patterns of nuclear population structure. Instead, extre- 1996), where deep burial partly facilitated by ants allows mely high chloroplast haplotype diversity, moderate to high longevity of seeds through protection from intense fires nuclear allelic diversity, IBD and low nuclear population (Gibson et al., 2011). The maintenance of germinability at

80 Journal of Biogeography 43, 70–84 ª 2015 John Wiley & Sons Ltd Phylogeographical structure of a fire-ephemeral vine

(a) (b)

(c)

Figure 4 Nuclear population genetic structure and diversity within Kennedia coccinea subsp. coccinea from south-western Australia derived from analysis of 11 microsatellite loci in 20 samples from 22 populations. (a) Pie charts represent mean relative ancestry of each population to each of five tess clusters, overlaid on a geographical map of sampling locations; (b) UPGMA tree, branch lengths represent Nei’s (1978) genetic distance. Relative ancestry to five tess clusters is represented by pie charts at node ends. The size of pie charts are proportional to mean allelic richness; (c) Mantel test showing significant correlation between genetic and geographical distance between population pairs. various depths reflects the potential for post-fire emergence forest trees with continuous ranges (Wheeler et al., 2003; and re-establishment from variously aged soil layers, enhanc- Nevill et al., 2014). In addition to a persistent seed bank, ing the opportunity for rare, diverse alleles to periodically effective movement of pollen by generalist pollinators emerge. These factors, together with variation in fire fre- throughout the intervening unburnt landscape will also facil- quency, size and intensity throughout the forest (depending itate and maintain nuclear genetic connectivity between on heterogeneous fuel load and ignition sources), would spatially isolated post-fire population patches. contribute to a potentially unique mosaic of diversity being elicited from the seed bank, both within and among CONCLUSIONS populations. In addition to diversity, a persistent seed bank is also Fire, and an obligate seeding life history, may drive phylo- expected to promote low levels of among-population differ- geographical patterns in fire-ephemeral species that deviate entiation by maintaining a high effective population size, from those otherwise expected for perennial species occupy- despite small above-ground population size or fluctuation ing the same biogeographical region. We found strong sig- (Honnay et al., 2008). Indeed, nuclear population differentia- nals of demographic expansion in K. coccinea that are likely tion in K. coccinea was lower than that observed for other to result from both a fire-ephemeral life history and a persis- non-fire-responsive herbaceous species in the jarrah forest tent soil seed bank, and that local fire frequency may be pos- (Coates et al., 2003; Hufford et al., 2012), but was surpris- itively associated with chloroplast haplotype diversity. Most ingly comparable with levels observed in widespread eucalypt notably, the predicted effects of genetic drift in a strongly

Journal of Biogeography 43, 70–84 81 ª 2015 John Wiley & Sons Ltd D. Bradbury et al. patchy, fire-driven system appeared to be buffered by a large, Sniderman, J.M.K., Sunnucks, P. & Weston, P.H. (2011) long-lived soil seed bank, which maintained, and possibly Decline of a biome: evolution, contraction, fragmentation, enhanced, total genetic diversity, and contributed to popula- extinction and invasion of the Australian mesic zone biota. tion connectivity together with ample pollen dispersal. While Journal of Biogeography, 38, 1635–1656. this research has focused on a single-species case study, fur- Chapuis, M.-P. & Estoup, A. (2007) Microsatellite null alleles ther research will benefit from a direct comparison of multi- and estimation of population differentiation. Molecular ple related species, for example by contrasting fire ephemeral Biology and Evolution, 24, 621–631. with perennial life-histories, in the same fire-prone environ- Chen, C., Durand, E., Forbes, F. & Francßois, O. 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(2013) Climate, not Appendix S1 Analysis of nuclear genetic structure of 22 Aboriginal landscape burning, controlled the historical Kennedia coccinea subsp. coccinea populations. demography and distribution of fire-sensitive conifer pop- Appendix S2 Values of Tajima’s D, Fu’s F and Ramos- ulations across Australia. Proceedings of the Royal Society B: S Onsins and Rozas’ R , excluding indels, for each separate Biological Sciences, 280, 2013–2182. 2 chloroplast locus. Schoknecht, N., Tille, P. & Purdie, B. (2004) Soil-landscape Appendix S3 Genetic characteristics of 11 nuclear mapping in south-western Australia: overview of methodol- microsatellite loci used to genotype Kennedia coccinea subsp. ogy and outputs. Resource Management Technical Report coccinea. 280. Department of Agriculture, Government of Western Australia. BIOSKETCH Segarra-Moragues, J.G. & Ojeda, F. (2010) Postfire response and genetic diversity in Erica coccinea: connecting popula- All authors are engaged in a broad research team investigat- tion dynamics and diversification in a biodiversity hotspot. ing patterns of plant phylogeography and population genetics 64 – Evolution, , 3511 3524. in the biodiversity hotspot of south-western Australia and Shaw, J., Lickey, E.B., Schilling, E.E. & Small, R.L. (2007) beyond, with a strong focus on the application of this Comparison of whole chloroplast genome sequences to research to conservation. choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. American Jour- Author contributions: D.B. analysed the nuclear data and nal of Botany, 94, 275–288. parts of the chloroplast data, interpreted the results and led Tajima, F. (1989) Statistical method for testing the neutral the writing. S.T. analysed the chloroplast data and con- mutation hypothesis by DNA polymorphism. Genetics, tributed to interpretation, figures and writing. S.M. and 123, 585–595. M.H. generated the nuclear and chloroplast data respectively. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. M.B. and D.C. designed the study, collected samples, inter- & Kumar, S. (2011) MEGA5: Molecular Evolutionary preted the results and contributed to the writing. Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Editor: Malte Ebach Biology and Evolution, 28, 2731–2739. Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUS- TAL W: improving the sensitivity of progressive multiple

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