Chloroplast and nuclear DNA exchanges among Begonia sect. Baryandra species (Begoniaceae) from Palawan Island, Philippines, and descriptions of five new species

The Philippine island of Palawan is highly biodiverse. During fieldwork there in 2011 & 2014 we found five unknown species in the large genus Begonia. The species are similar in their rhizomatous stems, four-tepaled flowers, inferior two- or three-locular ovaries with bilamellate placentas, and are assignable to Begonia sect. Baryandra. Our observations support the recognition of these as five new species endemic to Palawan: B. elnidoensis, B. gironellae, B. quinquealata, B. tabonensis and B. tenuibracteata which are described here. The five new species were added to phylogenies based Bayesian analysis of nrDNA (ITS) and chloroplast DNA (ndhA, ndhF-rpl32, rpl32-trnL, trnC-trnD), along with 45 other allied ingroup species. A majority of the species show incongruent positions in the two phylogenies, with evidence of prevalent chloroplast capture. Models show chloroplast capture is more likely in plant populations with high levels of inbreeding following a reduction in selfing rate after hybridisation; we suggest that this is a possible explanation for the massive amount of chloroplast exchange seen in our phylogeny, as Begonia species often exist as small isolated populations and may be prone to inbreeding depression. Our data also indicate a level of nuclear genetic exchange between species. The high prevalence of hybrid events in Begonia is potentially an important factor in driving genomic change and species evolution in this mega-diverse genus.


Introduction
Palawan is the only island province in the Philippines with more than 50% of its forest cover intact and it has been estimated that the island contains 1,522 species of flowering plants with PLOS  15-20% endemism [1]. The island is key not only as a conservation area, but also as a biogeographic nexus of great influence in the evolution of the eastern Malesian biota [2][3][4]. Recently our knowledge of the herbaceous diversity of the island has been augmented by the discovery of 11 new species of Begonia L. [5][6][7][8], nearly tripling the original number known to a total of 17 species. Of these, only Begonia mindorensis Merr. is distributed in both Luzon and Palawan; all the other 16 species are endemic to Palawan [9]. During recent fieldwork to the municipalities of El Nido and Quezon and Puerto Princesa City on Palawan, the authors found five species of Begonia which did not correspond to known taxa. Based on a study of relevant literature, type collections, as well as observation of living plants both from the field and in the greenhouse, we confirm that the five unknown Begonia are new species endemic to Palawan, which are here described and illustrated. They belong to Begonia sect. Baryandra A.DC., a section of the genus which has its centre of diversity in the Philippines [10]. Species in this section are rhizomatous, stemless, monoecious herbs, often lithophytic and associated with riverine habitats and waterfalls. The group arrived in the Philippines in the late Miocene, via long-distance dispersal from western Malesia and a point of entry likely to be in the northwestern region of the archipelago [3]. Palawan, Luzon, and Panay all bear early-branching lineages from this initial colonization [3]. The discovery of further diversity of the species rich genus Begonia on Palawan presents an opportunity to investigate the dynamics of the evolution of the highly endemic flora of the island.
Begonia is a mega-diverse genus of 1839 species [9], with a very high proportion of microendemics and hotspots of diversity in the Andes and Southeast Asia. This pattern of diversity begs an explantion; it has been suggested that the emergence of new habitats during the uplift of the Andes and the rapid geological evolution of the Malesian region has driven the diversification [11,12]. During exposure to new habitats, Begonia may be predisposed to forming new, narrowly endemic species due to remarkably low levels of gene flow, leading to weak selection pressures being able to shape new taxa in the absence of genetic contact with other populations [13]. Diversification may be further driven by as yet poorly understood characteristics of the Begonia genome, which may have an underlying genetic instability as evidenced by the highly variable chromosome number in the genus. This is suggestive of hybridisation followed by polyploidisation, with frequent chromosome fission and fusion leading to the observed dysploid variation in chromosome number in Begonia [14].
The extent of hybridisation in Begonia, at least in some clades, is only starting to become apparent from phylogenetic evidence. A majority of previous phylogenetic studies of Begonia have relied on a single genome for their data, usually the chloroplast [8,11,12,15] or nrITS [10,[16][17][18][19]. Some studies have analysed combined datasets, using nrITS and various chloroplast loci [20][21][22][23][24]. Some of these studies did highlight incongruence between the combined loci, either significant incongruence according to an ILD test [22] or topological but unsupported or moderately supported incongruence [20,24]. Only two studies did not uncover any significant incongruence between nuclear and chloroplast datasets [21,23]; however these both used the trnL intron from the chloroplast which tends to give low resolution in Begonia phylogenies. Two studies have presented phylogenies from two different genomes which showed considerable amounts of supported phylogentic incongruence in Begonia. Goodall-Copestake et al. [25] found incongruence between the chloroplast and mitochondrial phylogenies in a genus-wide study, and Hughes et al. [3] between nrITS and chloroplast phylogenies in a study of Begonia sect. Baryandra. It is this latter study to which we added further sampling of the new species mentioned above, and the further incongruence we uncovered prompted this manuscript.
Here we investigate the phylogenetic relationships of the five new Begonia species from Palawan and their relatives in Begonia sect. Baryandra using DNA sequence markers from the chloroplast genome and the nuclear genome. The chloroplast genome is maternally inherited in Begonia [26], whereas the markers we use from the nuclear genome (nuclear ribosomal DNA internal transcribed spacers (nrDNA ITS)) are diploid and bi-parentally inherited, and are located in the 45S ribosomal RNA genes. The few previous studies which have demonstrated phylogenetic incongruence between various genomic markers in Begonia hint that hybridisation has the potential to be important in the generation of the massive diversity of the genus. The markers from different genomes allow us to gain further insight into the prevalence of past hybridisation events in the evolution of the species in Begonia sect. Baryandra.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants [27], and hence the new names contained in the electronic publication of a PLoS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to International Plant Name Index (IPNI), from where they will be made available to the Global Names Index. The IPNI Life Science Identifiers (LSIDs) can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS, and The Begonia Resource Centre (http://padme.rbge.org.uk/begonia/).

Molecular phylogenetics
Molecular phylogenies were constructed based DNA sequences from the chloroplast genome (four noncoding regions ndhA intron, ndhF-rpl32 spacer, rpl32-trnL spacer, trnC-trnD spacer) and the nuclear genome (nuclear ribosomal internal transcribed spacers ITS1 and 2 with the 5.8S gene). Taxon sampling was based on that of Hughes et al. [3], supplemented with 14 new accessions including all the new species described herein. The ingroup, Begonia sect. Baryandra, has been found to be strongly supported as monophyletic [3,10]; the outgroup sampling for both phylogenies matches that of Hughes et al. [3]. A total of 91 accessions were sampled for the chloroplast regions, representing 67 taxa including outgroups, and 69 accessions representing 50 taxa (including outgroups) for the nuclear region. Voucher and GenBank accession information is listed in S1 Table. The DNA extraction, PCR and sequencing were carried out as in Hughes et al. [3]. The following bases were excluded from the chloroplast alignment because of missing data at the ends of regions, or alignment uncertainty due to long mononucleotide repeats: 1-10, 1390-1540, 2510-3050, 4530-4645, 4746-4910, 4943-4997, 5720-5810, 6970-7125, 8050-8089; and from the nuclear alignment bases 494-548 were excluded. Appropriate models of DNA sequence evolution for the aligned datasets were assessed with the program jModeltest 2.1.3 [28] using the corrected Akaike information criterion (AICc) to estimate the model with the closest fit to the data. For both the chloroplast and nuclear alignment, the GRT+G+I model was the most probable (AIC weight 1.00 and 0.66 respectively).
Bayesian phylogenetic analyses were carried out separately on the chloroplast and nuclear data sets using the program MrBayes 3.2.6 [29]. Each data set was treated as a single partition, analysed under the appropriate model of sequence evolution and the default parameters of two runs with four chains each, run for 10 000 000 generations with a sample tree taken every 10 000 generations. The convergence of the MCMC chains of the two runs was assessed by inspection of the trace plots of parameters using Tracer ver. 1.6 [30]. The effective sample sizes (ESS) of all parameters were > 200, indicating that each parameter was sampled satisfactorily. The first 25% of sampled trees were discarded as burn-in, and the remainder summarized as a maximum clade credibility tree. Trees were visualised using the APE package [31]. Strict and semi-strict consensus trees combining the two analyses were constructed in PAUP [32].

Incongruence tests
Alignments with matching sampling for both chloroplast and nuclear markers were constructed for use in conducting an Incongruence Length Difference test (ILD) [33] and measures of internode certainty (IC) [34]. The ILD test was conducted using PAUP, using 100 replicates based on heuristic searches with TBR branch swapping, each with 10 random addition replicates and Maxtrees set to 100. In order to further examine topological incongruence between the datasets, post burn-in samples of trees resulting from the Bayesian phylogenetic analysis of the chloroplast and ITS data were combined into a single majority rule consensus (MRC) tree with IC values for each node using RaxML [35]. The IC metric (scaled between 0 and 1) represents the certainty for each internal branch, taking into account the frequency of the most prevalent conflicting bipartition in the population of trees used to make the consensus. For example, if there are two conflicting bipartitions between the chloroplast and ITS derived topologies, each present at 100% frequency in the different datasets, then IC = 0, reflecting the complete incongruence between the genomes. A bipartition with 100% frequency in trees derived from the chloroplast dataset and 50% frequency in the ITS dataset, contradicted by 50% of the remaining trees in the ITS dataset would have an IC = 0.18. Comparing all post burn-in trees allows us to further investigate the full range of topologies produced by the two separate analyses, rather than just comparing a separate consensus of each.

Molecular phylogenetics
The ITS alignment consisted of 853 bp in length, with 299 of these being parsimony-informative. The chloroplast alignment consisted of 6764 bp in length, with 370 of these being parsimony informative. A majority of the species sampled in our Begonia sect. Baryandra phylogenies show some incongruence between their placements in the chloroplast and nuclear phylogenies (Fig 1 and S1 Fig). No readable ITS sequence could be obtained for the terminal B. hernandioides1, as the electropherograms showed polymorphisms at most sites. The ITS electropherograms for terminals B. nigritarum3, B. tabonensis, B. wadei, B. woodii1 and B. woodii2 showed some polymorphism towards the end of the reads, however good sequence data was obtained for all the alignment for these taxa with the exception of portions of the 5.8S gene and the ITS2 spacer.

Incongruence tests
The ILD test, as expected, showed significant incongruence between the chloroplast and ITS datasets (P = 0.01). Only two clades of more than three taxa were present in the MRC tree Given the amount of incongruence between the phylogenies, assessing which species have a similar position in the two analyses is difficult. A large number of species show hard incongruence between our analyses, being in conflicting positions with support of PP >0.95. We split these into two categories, depending on the spread of the differing positions in the two analyses. Firstly, there are species where the incongruence could potentially be due to lineage sorting, as the branch lengths are relatively short and only one node is involved.

Discussion
The five new species described here show that Palawan is biodiverse and underexplored; four of the species are from nearby localities in the El Nido municipality, and so further exploration in other parts of the island is likely to reveal additional novelties. The ongoing discovery of biodiversity on Palawan highlights the need to preserve its current forest cover and ensure protected areas are sustainably managed. The declaration by the Palawan Council for Sustainable Development (PCSD) of the Cleopatra's Needle Critical Habitat [36] status is particularly welcome. This is the largest area of critical habitat designation in the Philippines and a landmark for conservation in the region.
The evolution of the Begonia diversity on Palawan is a consequence of relict lineages from the late Miocene combined with recent Pliocene-Pleistocene diversification [3]. The increase in sampling in this study has shed further light on the phylogenetic relationships of Palawan Begonia, and has highlighted remarkable levels of incongruence between nuclear and plastid derived phylogenies. Incongruence between plastid and nuclear phylogenies can be attributed to either incomplete lineage sorting or chloroplast capture [37]. Incomplete lineage sorting is the inheritance of alleles during the speciation process whose genealogy differs from the bulk of the genome which follows the species phylogeny. It is a phenomenon which occurs between recently or previously rapidly diverged species. Chloroplast capture occurs when the chloroplast genome is introgressed from another species, leading to a genealogical mismatch to the nuclear genome. Markers from the nuclear genome would give the correct species tree, whereas chloroplast markers would reflect the phylogenetic position of the plastid donor. Chloroplast capture can happen between either recently diverged or more distantly related species [38].
We estimate that approximately half of the 49 species in our phylogeny show phylogenetic incongruence which is likely to be due to hybridisation rather than stochastic lineage sorting, owing to the deep incongruence shown in the different gene trees. Chloroplast capture is expected to be more commonly observed than nuclear gene exchange in plants, due to the smaller effective population size of the haploid plastid genome, making it more likely that a foreign chloroplast genotype will become fixed in populations of the recipient species [39]. Our phylogeny demonstrates that in Begonia sect. Baryandra prevalent chloroplast capture seems to be the case; for example, the three samples of B. gironellae are monophyletic in the nrDNA phylogeny, whilst appearing in two positions in the chloroplast phylogeny (Fig 1); the plastid genome of B. gironellae3 is likely to be captured from a different lineage. Also, B. quinquealata appears in a clade of species with straight fruit wings (e.g., Fig 2I) in the chloroplast phylogeny, whereas in the rDNA phylogeny it sits morphologically comfortably in a grade with several species sharing markedly cucullate fruit wings (e.g., Fig 3H), and hence the rDNA represents the most probable species tree. Begonia palawanensis and B. tenuibracteata share a very similar chloroplast haplotype, yet they belong to different sections of the genus (sects. Petermannia and Baryandra respectively). The ITS sequence data from B. palawanensis places it outside the outgroups used for the analysis, and in a clade reflecting its morphology and taxonomy, with other members of Begonia sect. Petermannia from Borneo, the Philippines and New guinea (analysis not included here). The samples for these two species were taken from nearby localities near Salakot Falls, and it is probable that the population of B. palawanensis has captured the chloroplast from adjacent populations of B. tenuibracteata. Hence it seems that a large proportion of the 49 species we sampled from Palawan have acquired a foreign plastid at some point during their evolution. Chloroplast capture may be aided in Begonia by the very weak barriers to hybridisation in the genus, with F 1 hybrids being readily made between even distantly related species [14]. However, natural F 1 hybrids in Begonia have frequently been found to be pollen sterile [40,41] and even crosses between isolated populations of the Mexican species B. heracleifolia have been found to have reduced pollen fertility [42]. Despite these observed barriers to hybrid fertility, in order to explain the prevalence of chloroplast capture it seems likely that in many cases it infers a fitness benefit. Begonia species exist in isolated populations connected by only low levels of gene flow [13,43,44], potentially leading to inbreeding depression. A model of chloroplast capture by Tsitrone et al. [38] demonstrates that capture is facilitated when the selfing rate is reduced in hybrids (for example by reduced pollen fertility) in populations with strong inbreeding depression. This would lead to increased introgression from pollen with the 'native' genotype, with potentially a relative increase in female fitness in individuals with a foreign chloroplast genotype. Most Begonia species are fully self-compatible and partially selfing [13,42]; if inbreeding depression is significant in the genus, this could explain the high degree of chloroplast capture we have observed in Palawan Begonia. The very high levels of population isolation observed in Begonia also mean that chloroplast capture is more likely from a neutral perspective than in more panmictic groups, due to the reduced flux of genes from other populations which could 'flush out' foreign haplotypes [45]. Hybridisation seems to be linked to geographic proximity, as in the cases of B. palawanensis and B. tenuibracteata described above, of B. elnidoensis and B. mindorensis which have overlapping distributions in northern Palawan, and of the within-Palawan hybridisation evidenced by the other new species described here. This scenario is congruent with the geographically limited gene flow recorded for Begonia species [13]. There is no evidence for morphological intermediacy in the species observed to have undergone past hybridisation; for example B. elnidoensis is morphologically and ecologically very divergent from B. mindorensis, and the three individuals of B. gironellae with differing phylogenetic histories are not morphologically dissimilar. We found no evidence for morphologically intermediate F1 hybrids in the field. Some of the incongruence observed is likely to be due to nrDNA capture, as there are some species in which the chloroplast phylogeny reflects the most probable species tree in terms of morphology and distribution, with the nrDNA marker showing an incongruent placement. This is the case in Begonia wadei and B. elnidoensis, which are succulent stemmed species ecologically aberrant for the genus. Their placement at the base of a Palawan endemic clade which began diverging in the late Miocene [3] is much more plausible than their highly nested placement in a clade of rhizomatous forest-dwelling species predominantly from Luzon and Panay. The two divergent nrDNA genotypes possessed by B. tagbanua are very divergent; although potentially they could represent ancestral diversity, it is more plausible that the nrDNA genotype of B. tagbanua2 was captured from a different lineage.
The nrDNA genes are arranged in tandem repeats on regions of chromosomes known as nucleolus organiser regions (NOR). Nucleoli form within the nucleus around the NOR's, and are responsible for ribosome production. The number of major NOR loci in plants has been found to vary from 1 to 32, although a majority have one or two [46]. DNA sequences of the ribosomal genes and their associated spacers are generally treated as a single locus in the context of phylogenetic analysis, due to concerted evolution homogenising the sequences within and between loci [47]. If we assume Begonia is typical amongst angiosperms in having one or two NOR loci, it means that we have detected nuclear genome exchange in at least two species on Palawan using a marker on just one or two chromosomes. Hence it would seem either (i) the NOR loci are preferentially inherited or selected for during hybridisation events, or (ii) hybridisation has been rampant among Palawan Begonia; the latter would seem more likely. The forces driving the large amount of chloroplast exchange between Begonia species on Palawan seems also to have driven nuclear gene exchange.
Our sampling of a single sequence to represent the likely one or two NOR loci present in Begonia is a tiny snapshot of the nuclear genome. If this miniscule sampling is able to identify a signal of nuclear genome exchange between species, it is highly likely to be much more prevalent than we detect here. Previous studies have emphasised how allopatry and absence of gene flow have been important drives of diversification in Begonia [13,22,44]; however, it seems that ongoing genetic exchange between species is more prevalent than suspected, and begs further investigation. Next generation phylogenomic approaches are needed to reveal how much of a patchwork the nuclear genome of Begonia species is, and to identify the scale of betweenspecies genetic exchange from the level of the single gene to entire chromosomes. This may reveal insights into why Begonia is so adept at generating new species.
Given the phylogenetic chaos we have uncovered in Begonia sect. Baryandra, it may seem difficult to pin down a species concept, and certainly a monophyletic species concept is out of the question. However, our taxonomists species concept continues to serve us and other Begonia taxonomists well; in the last 2 years 147 new species have been described [9]. Even in the light of molecular phylogenetic data, it seems that careful observation of plants in the field and herbarium remain our most faithful tools for describing the basic units for conservation and research in Begonia.

Taxonomic treatment
Key to Begonia sect. Baryandra on Palawan. As many Begonia species are narrow-range endemics, we have added distribution information to help confirm determinations and also highlight new records for other areas (Table 1). Types and representative specimens for most species are available online [9]. Monoecious, thick-stemmed herb. Stem unbranched, ascending or pendent from limestone rock face, to 50 cm or longer, to 3 cm thick, internodes 0.5-3 cm long. Stipules persistent, pale  (Fig 6). The type specimen was collected from El Nido, and it also occurs in Lagen and Miniloc Islands and other islands and islets adjacent to El Nido as observed by some of the authors during an expedition in 2011. After observation of many individuals in different locations, it is clear that this species is potentially immortal through continuous basal emergence of new stems. Considering the leaf scars and internode length, there are alternate sequences of short and long internodes, more pronounced on individuals growing in partly shaded situations, due to the strong tuberisation Evolution and five new species of Palawan Begonia of the stem in individuals growing on fully exposed sea cliffs making the differences less obvious. About 10 to 12 leaves are produced between long (1.5 to 2 cm long) internodes, and about the same between short internodes (0.5 to 1 cm long). This corresponds to faster and slower growth in the wet and dry season on Palawan. Stem elongation appears to be 20 to 30 cm annually, the longest stems reaching about 120 cm, which are probably four to six years old Evolution and five new species of Palawan Begonia and likely represent the maximum age. New stems emerge from one of the first short internodes at the base of living older stems and they produce adventitious roots for fixation and nutrition. Flowering seems to be continuous all the year round, as observed from the couple of scars at each internode; one scar for the petiole and just above one scar for the inflorescence peduncle. Thus this species flowers both in rainy and dry seasons. During fruit maturation, the main inflorescence axis remains turgescent and alive, whereas the secondary axes dry out but remain attached. This junction permits the dry, dehisced capsules to freely shake in the wind. The authors have observed active anemochory in this species.
Provisional conservation assessment. This species does not depend on a forest habitat, as it grows crevices in coralline sea cliffs. This habitat is already exposed and hence not under threat from forest clearance, and is also relatively inaccessible, and so is not at great risk of increased disturbance. Given there are a number of populations around coastal northern Palawan, and the fact the species was observed growing near a rubbish dump in El Nido town, we consider B. elnidoensis to belong the Least Concern IUCN category [48].
Distribution and ecology. Endemic to Tanabag, Puerto Princesa in northern Palawan, the Philippines; occurring on riverbanks or steep mossy slopes at ca. 30 m elevation.
Provisional conservation assessment. Two populations were observed by the authors at the type locality at the lower reaches of the Tanabag River, one of which was locally abundant. The species is very likely to grow further up the meandering and relatively inaccessible riverbanks. If further populations exist upstream, the species could belong to the Least Concern category, or if the populations we observed are the only ones existing then VUD2 would be appropriate. Given this uncertainty we assign B. gironellae to the Data Deficient category.
Etymology. The species is named in honor of Prof. Elizabeth P. Gironella of the Palawan State University, who accompanied and guided us during the field trip to the type locality and southern Palawan.
Distribution and ecology. Begonia quinquealata is endemic to Palawan. On low vertical cliff face or rock-strewn at base of cliffs, broadleaved forest margin.
Provisional conservation assessment. The species exists as a single population at the type and only locality, with ca. 100 individuals in an area several metres square. Searches around the area failed to locate further individuals. The population is near a drainage channel along a roadside, where the overhanging vegetation is being cleared. We consider Begonia quinquealata to belong to the Critically Endangered category, under criteria CRB2abiii&v (area of occupancy <10km2, a single location, and a continuing decline in the area and quality of habitat, and the number of mature individuals).
Distribution and ecology. Begonia tabonensis is endemic to Lipuun Point, north of Quezon municipality, in southwestern Palawan Island. It occurs on semishaded limestone cliffs around Tabon Cave, an archeological and natural heritage site, in lowland forest at ca. 30 m elevation. Provisional conservation assessment. The species is common at the type and only locality of the Tabon Cave vicinity. The site is visited by tourists, but is sustainably managed by the local community and the National Museum Palawan Branch. Although it has a small distribution, we consider B. tabonensis to belong to the IUCN cateory Least Concern as long as the site remains undisturbed.
Etymology. The specific epithet refers to Tabon Cave where the new species was discovered.
Distribution and ecology. Begonia tenuibracteata is endemic to central Palawan, occurring on mossy boulders along road cut in shaded, wet lowland forest, near Salakot Falls, Napsan.
Provisional conservation assessment. The species is locally abundant at the type and only locality, however the number of individuals in the Salakot Falls area has decreased significantly Evolution and five new species of Palawan Begonia according to our observations at the site in 2006 and 2011. We consider B. tenuibracteata to belong to the IUCN category Endangered (ENC1) due to this observed decline.
Etymology. The specific epithet refers to the hyaline, membranaceous bracts of this new species. Evolution and five new species of Palawan Begonia Notes. Begonia mindorensis, a widespread species in lowland to montane forests in Luzon, Mindoro and Palawan, also produces the unusual, conspicuous, persistent bracts on the inflorescences like B. tenuibracteata. However, B. tenuibracteata is sharply distinct by the ovate to lanceolate (vs. widely to depressed ovate) bracts that are hyaline, membranaceous (vs. coriaceous), glabrous or with very sparse sessile glands (vs. densely clothed with sessile glands). In addition, B. tenuibracteata differs by the congested rhizomes with internodes only to 3 mm (vs. 20 mm) long; shorter petioles (to 7 cm vs. 10-25 cm long); velvety (vs. glossy) leaf upper surface; shorter inflorescence (to 22 cm vs. over 35 cm long); and fewer stamens (40-50 vs. ca. 70).
Supporting information S1 Table. Genbank accession numbers and voucher information for the DNA sequences used in the phylogenetic analyses. Samples newly sequenced for this study are highlighted with an asterisk. (PDF)