 phylogenetic informativeness of GBS data sets, so researchers are encouraged to consider low sample coverage thresholds when inferring phylogenies. We also found that setting a strict filtering level for the number of SNPs in a locus (to remove poorly aligned or repetitive regions) removed good loci and phylogenetic signal. While a small number of SNPs per locus is a reasonable filter for studies on one or two species, it can be detrimental to phylogenomic studies with multiple divergent species and outgroups. In our study system, we found that loci in one species might differ from another at a few positions, but differ from a different relative at other positions, often resulting in many SNPs (and phylogenetically informative positions) recovered per locus. As such, researchers working in similarly multi-species systems would benefit from not setting strict limits on the number of SNPs in a final locus.
Our GBS data provide a first measure of genomic variation in the Triodia basedowii species complex and new insights into the distinction of taxa and their evolutionary relationships. There is a close correspondence between genomic entities and a priori taxon identifications, across large geographical ranges and even where taxa co-occur, consistent with the hypothesis that these taxa are distinct species.
Well-supported clades recovered in phylogenetic and species tree analyses are partly congruent and incongruent with those found in Anderson et al. [46]. In some cases, GBS results provide support where previous results were ambiguous. For example, Anderson et al. [46] hypothesised a sister relationship between T. "Shov" and T. "War" based on morphology and the presence of shared ITS copies, but found no or ambiguous support for this in molecular phylogenies using ITS/ETS and a chloroplast marker. Our GBS analysis strongly supports this sister relationship, with phylogenetic and species trees always recovering it and clustering analyses showing the two entities to be close but distinct. Similarly, Anderson et al. [46] were unable to resolve T. "LSandy" as distinct from T. basedowii despite morphology and field observations suggesting that it is. In our GBS trees, T. "LSandy" and T. basedowii were consistently recovered as sisters, with clustering analyses and branch lengths in concatenation and MP-EST trees suggesting substantial divergence between them. Anderson et al. [46] also found insufficient evidence to recognize the putative taxon T. "shovb" as distinct from T. lanigera, and the ITS/ETS analysis placed the combined taxon as part of a polytomy with T. basedowii. Our GBS results consistently recover their close relationship and limited divergence, supporting the former conclusion (we continue to treat them as divergent populations of a single species), but place them as sister to T. "shova" rather than T. basedowii. This agrees with chloroplast data [46], suggesting that the ITS result may represent introgression from T. basedowii.
The new putative taxa T. "nana" and T. "Panna" were consistently distinct across analyses but unstably placed in the GBS trees. Branch lengths subtending these two taxa are consistently long (especially for T. "Panna"), suggesting that these morphologically distinct entities should be recognised as distinct new species in the complex. T. "Panna" is of particular importance as it is known from a single population, and, despite being a tetraploid, has the lowest heterozygosity for taxa in our study based on statistics from the PyRAD assembly. This suggests that the population has experienced a severe bottleneck, which together with its restricted distribution makes it of conservation concern.
Ploidy variation in the Triodia basedowii complex does not follow taxonomic boundaries, suggesting that most polyploid events represent sporadic cases of autopolyploidy arising recently within species. Our GBS analyses show that the widespread T. basedowii forms a distinct genomic cluster, with some distinction between populations but without tetraploid populations clustering distinctly from diploid populations (e.g. central Australian populations cluster together despite different ploidy levels). Multiple ploidy levels are also found in T. lanigera (including some of its T. "shovb" populations) and in a population of putative hybrids between tetraploid T. lanigera and diploid T. "shova", suggesting that differing ploidy is not acting as a strong reproductive barrier in this case. The co-occurrence of individuals with different ploidy (as in e.g. a population of T. "wcoast") is rare in the complex, however, suggesting some segregation between ploidy levels may occur. Outside the complex, indirect evidence in the form of heterozygosity statistics for PyRAD loci (elevated in tetraploids in the unmerged data) suggest there may be diploid plants in the tetraploid T. concinna population. These results suggest that polyploidy has not been a major speciation driver in the complex.
Our species tree analyses are helpful in assessing the distinction of entities that are dispersed or not sister in the trees (e.g. T. "Shov" from T. basedowii), but because we designated taxa a priori, these analyses do not help to assess whether sisters are distinct (e.g. T. "Peed" from T. "wcoast"). Our decision to recognise taxa in these cases depends on assessing the degree of genomic divergence (from clustering analyses and phylogenetic branch lengths) along with supporting evidence (from e.g. morphology or distribution). For example, the geographically disjunct putative taxa T. "Peed" (diploid) and T. "wcoast" (tetraploid/diploid) have some morphological distinction but are indistinguishable in ITS/ETS [46]. Our GBS results suggest moderate divergence between the two entities, and they do not always form a single clade (e.g. paraphyletic in Fig 4B, S13B and S14B Figs), but our sampling of T. "wcoast" is relatively poor. With these ambiguous results, we conservatively treat them as divergent populations of one species, pending more extensive sampling. This example contrasts with that of T. basedowii and T. "LSandy", where there is more clearly morphological distinction, a more substantial degree of genomic divergence, and co-occurrence without evident mixing.
Localities where putative taxa co-occur provide a useful test for species distinction and an opportunity to assess the frequency of hybridization. Anderson et al. [46] provided evidence of hybridization between some taxa in the T. basedowii complex, and our GBS results both support and contradict that evidence. GBS results from one population ("Amb") support previous evidence for hybridization between T. lanigera and T. "shova". Individuals with intermediate morphologies clearly form genomic clusters between the putative parental species in the PCoA (Fig 5B), with those identified as more like T. "shova" clustering closer to that taxon and similarly for T. lanigera. In contrast, a population ("mixSPH") containing both T. lanigera and T. "shova" that showed evidence of some ITS copy sharing (Table 6) did not show evidence of genomic mixing in our results. Multiple T. "shova" individuals possess both T. lanigera and T. "shova" ITS copies, while the same cannot be said for T. lanigera individuals, and our GBS results place all samples from this population with their respective taxa and without suggestions of genomic intermediacy.
Table 6. ITS copy types for co-occurring Triodia lanigera and T. "shova".
The contrasting results suggest that there is limited introgression of rDNA despite relatively strong barriers across the rest of the genome. Similarly, a population ("RoyH") of individuals found to possess ITS copies characteristic of T. "Shov" and T. "War" showed no evidence of genomic mixing in our results following resampling. Samples from the population clustered with T. "Shov", suggesting that introgression may be restricted to rDNA or that our second sampling for GBS (only four plants) comprised parental plants rather than hybrids, if the population contained both. In other populations with co-occurring taxa (e.g. "Roy1" comprising T. "Shov" and T. basedowii, with one probable misidentification, and "Nifty" comprising T. "War" and T. basedowii) there is no evidence of genomic mixing or rDNA introgression, which suggests these taxa are reproductively isolated.
By addressing GBS bioinformatic challenges, such as overlapping reads following imperfect size selection and paired-end read locus duplication, a biologically meaningful phylogenetic signal from across the genome can be extracted for study systems lacking prior genomic knowledge. The continuing improvement in next-generation sequencing read length holds promise for using analytical methods that rely on extracting phylogenetic signal from each locus, such as new species tree approaches. Here we have demonstrated their use with our assembled GBS loci and shown that they are more robust to variation in assembly parameters than commonly used concatenation approaches. Our GBS data and analyses have improved the resolution of relationships in the T. basedowii complex and provided new insights into processes influencing evolution of the group (e.g. polyploidy and partial introgression). These results will support an upcoming taxonomic revision of the complex, with the recognition of new species including one of conservation significance, and complement the findings of Anderson et al. [46] with regard to the high diversity of the complex in the Pilbara region of Western Australia. We encourage other researchers working in similarly difficult taxonomic systems to use the relatively affordable and accessible GBS approach outlined here for generating and analysing genomic data.
S1 File. Sample information for sequencing and analyses.
S2 File. Details of GBS Library Prepartion.
S3 File.