Randall L. Small

The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis

Chloroplast DNA sequences are a primary source of data for plant molecular systematic studies. A few key papers have provided the molecular systematics community with universal primer pairs for noncoding regions that have dominated the field, namely trnL-trnF and trnK/matK. These two regions have provided adequate information to resolve species relationships in some taxa, but often provide little resolution at low taxonomic levels. To obtain better phylogenetic resolution, sequence data from these regions are often coupled with other sequence data. Choosing an appropriate cpDNA region for phylogenetic investigation is difficult because of the scarcity of information about the tempo of evolutionary rates among different noncoding cpDNA regions. The focus of this investigation was to determine whether there is any predictable rate heterogeneity among 21 noncoding cpDNA regions identified as phylogenetically useful at low levels. To test for rate heterogeneity among the different cpDNA regions, we used three species from each of 10 groups representing eight major phylogenetic lineages of phanerogams. The results of this study clearly show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.

The tortoise and the hare: choosing between noncoding plastome and nuclear Adh sequences for phylogeny reconstruction in a recently diverged plant group

Phylogenetic resolution is often low within groups of recently diverged taxa due to a paucity of phylogenetically informative characters. We tested the relative utility of seven noncoding cpDNA regions and a pair of homoeologous nuclear genes for resolving recent divergences, using tetraploid cottons (Gossypium) as a model system. The five tetraploid species of Gossypium are a monophyletic assemblage derived from an allopolyploidization event that probably occurred within the last 0.5-2 million years. Previous analysis of cpDNA restriction site data provided only partial resolution within this clade despite a large number of enzymes employed. We sequenced three cpDNA introns (rpl16, rpoC1, ndhA) and four cpDNA spacers (accD-psaI, trnL-trnF, trnT-trnL, atpB-rbcL) for a total of over 7 kb of sequence per taxon, yet obtained only four informative nucleotide substitutions (0.05%) resulting in incomplete phylogenetic resolution. In addition, we sequenced a 1.65-kb region of a homoeologous pair of nuclear-encoded alcohol dehydrogenase (Adh) genes. In contrast with the cpDNA sequence data, the Adh homoeologues yielded 25 informative characters (0.76%) and provided a robust and completely resolved topology that is concordant with previous cladistic and phenetic analyses. The enhanced resolution obtained using the nuclear genes reflects an approximately three- to sixfold increase in nucleotide substitution rate relative to the plastome spacers and introns.

L. A. S. JOHNSON REVIEW No. 2 Use of nuclear genes for phylogeny reconstruction in plants

Molecular data have had a profound impact on the field of plant systematics, and the application of DNA-sequence data to phylogenetic problems is now routine. The majority of data used in plant molecular phylogenetic studies derives from chloroplast DNA and nuclear rDNA, while the use of low-copy nuclear genes has not been widely adopted. This is due, at least in part, to the greater difficulty of isolating and characterising low-copy nuclear genes relative to chloroplast and rDNA sequences that are readily amplified with universal primers. The higher level of sequence variation characteristic of low-copy nuclear genes, however, often compensates for the experimental effort required to obtain them. In this review, we briefly discuss the strengths and limitations of chloroplast and rDNA sequences, and then focus our attention on the use of low-copy nuclear sequences. Advantages of low-copy nuclear sequences include a higher rate of evolution than for organellar sequences, the potential to accumulate datasets from multiple unlinked loci, and bi-parental inheritance. Challenges intrinsic to the use of low-copy nuclear sequences include distinguishing orthologous loci from divergent paralogous loci in the same gene family, being mindful of the complications arising from concerted evolution or recombination among paralogous sequences, and the presence of intraspecific, intrapopulational and intraindividual polymorphism. Finally, we provide a detailed protocol for the isolation, characterisation and use of low-copy nuclear sequences for phylogenetic studies.

Biogeography and Floral Evolution of Baobabs Adansonia, Bombacaceae as Inferred From Multiple Data Sets

The phylogeny of baobab trees was analyzed using four data sets: chloroplast DNA restriction sites, sequences of the chloroplast rpl16 intron, sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, and morphology. We sampled each of the eight species of Adansonia plus three outgroup taxa from tribe Adansonieae. These data were analyzed singly and in combination using parsimony. ITS and morphology provided the greatest resolution and were largely concordant. The two chloroplast data sets showed concordance with one another but showed significant conflict with ITS and morphology. A possible explanation for the conflict is genealogical discordance within the Malagasy Longitubae, perhaps due to introgression events. A maximum-likelihood analysis of branching times shows that the dispersal between Africa and Australia occurred well after the fragmentation of Gondwana and therefore involved overwater dispersal. The phylogeny does not permit unambiguous reconstruction of floral evolution but suggests the plausible hypothesis that hawkmoth pollination was ancestral in Adansonia and that there were two parallel switches to pollination by mammals in the genus.

Rapid diversification of the cotton genus (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast genes

Previous molecular phylogenetic studies have failed to resolve the branching order among the major cotton (Gossypium) lineages, and it has been unclear whether this reflects actual history (rapid radiation) or sampling properties of the genes evaluated. In this paper, we reconsider the phylogenetic relationships of diploid cotton genome groups using DNA sequences from 11 single-copy nuclear loci (10 293 base pairs [bp]), nuclear ribosomal DNA (695 bp), and four chloroplast loci (7370 bp). Results from individual loci and combined nuclear and chloroplast DNA partitions reveal that the cotton genome groups radiated in rapid succession following the formation of the genus. Maximum likelihood analysis of nuclear synonymous sites shows that this radiation occurred within a time span equivalent to 17% of the time since the separation of Gossypium from its nearest extant relatives in the genera Kokia and Gossypioides. Chloroplast and nuclear phylogenies differ significantly with respect to resolution of the basal divergence in the genus and to interrelationships among African cottons. This incongruence is due to limited character evolution in cpDNA and either previously unsuspected hybridization or unreliable phylogenetic performance of the cpDNA characters. This study highlights the necessity of using multiple, independent data sets for resolving phylogenetic relationships of rapidly diverged lineages.

Chloroplast DNA phylogeny and phylogeography of the North American plums (Prunus subgenus Prunus section Prunocerasus, Rosaceae)

The North American plums are a closely related group that are not easily circumscribed, have overlapping morphologies, and are known to hybridize. We previously showed that the North American plums are a closely related, monophyletic group of taxa with little to no cpDNA sequence divergence between taxa. In that study, we came to the unanticipated conclusion that relationships inferred among the taxa contrast sharply with previously defined groups based on morphological characters. Here the aim was to determine if the primary cpDNA haplotypes identified in our earlier study are confined to the taxa in which they were initially observed. The cpDNA rpL16 intron was sequenced for 207 accessions of the 17 North American plum taxa plus Prunus texana. The results show that many taxa contain more than one of the three primary cpDNA haplotypes. Aside from the results found in sect. Prunocerasus, this study has broader implications for phylogenetics in general. The common practice of choosing a single exemplar to represent a taxon can be profoundly misleading in closely related groups. In hindsight, the possibility existed in our earlier study that we could have chosen a different combination of exemplars, which could have resulted in a different inferred phylogeny.

CRYPTIC REPEATED GENOMIC RECOMBINATION DURING SPECIATION IN GOSSYPIUM GOSSYPIOIDES

Abstract The Mexican cotton Gossypium gossypioides is a perplexing entity, with conflicting morphological, cytogenetic, and molecular evidence of its phylogenetic affinity to other American cottons. We reevaluated the evolutionary history of this enigmatic species using 16.4 kb of DNA sequence. Phylogenetic analyses show that chloroplast DNA (7.3 kb), nuclear ribosomal internal transcribed spacers (ITS; 0.69 kb), and unique nuclear genes (8.4 kb) yield conflicting resolutions for G. gossypioides. Eight low-copy nuclear genes provide a nearly unanimous resolution of G. gossypioides as the basalmost American diploid cotton, whereas cpDNA sequences resolve G. gossypioides deeply nested within the American diploid clade sister to Peruvian G. raimondii, and ITS places G. gossypioides in an African (rather than an American) clade. These data, in conjunction with previous evidence from the repetitive fraction of the genome, implicate a complex history for G. gossypioides possibly involving temporally separated introgression events from genetically divergent cottons that are presently restricted to different hemispheres. Based on repetitive nuclear DNA, it appears that G. gossypioides experienced nuclear introgression from an African species shortly after divergence from the remainder of the American assemblage. More recently, hybridization with a Mexican species may have resulted in cpDNA introgression, and possibly a second round of cryptic nuclear introgression. Gossypium gossypioides provides a striking example of the previously unsuspected chimeric nature of some plant genomes and the resulting phylogenetic complexity produced by multiple historical reticulation events.

Addressing the “hardest puzzle in American pomology:” Phylogeny of Prunus sect. Prunocerasus (Rosaceae) based on seven noncoding chloroplast DNA regions

Prunus subg. Prunus sect. Prunocerasus (Rosaceae) is a North American taxon with 17 commonly recognized taxa. To test the hypothesis of monophyly for the section we sequenced the trnG and rpL16 introns and the trnH-psbA and trnS-trnG intergenic spacers for at least two representatives of each of the five subgenera in Prunus. Additionally we sampled heavily among Prunus subg. Prunus sections Prunus and Armeniaca and Prunus subg. Amygdalus because these groups are putatively most closely related to Prunocerasus. Once monophyly of sect. Prunocerasus was shown we added the sequences of trnL and rpS16 introns and the trnL-trnF spacer in an attempt to increase resolution within the section. The species of sect. Prunocerasus showed an initial split with P. subcordata, the only species from western North America, sister to the rest of the group. The remaining species fell into three primary clades. Within each of the three primary clades there was little phylogenetic resolution. Lastly, we present evidence that P. texana, previously classified in subg. Amygdalus, may be a plum or at least contain a Prunocerasus chloroplast. This is the first phylogenetic hypothesis presented for sect. Prunocerasus, and the clades recovered contrast sharply with previously defined groups based on morphological characters.

Phylogeny of Hibiscus sect. Muenchhusia (Malvaceae) Based on Chloroplast rpL16 and ndhF, and Nuclear ITS and GBSSI Sequences

Abstract Hibiscus section Muenchhusia is a North American taxon with five species as recognized in the most recent taxonomic revision: H. coccineus, H. dasycalyx, H. grandiflorus, H. laevis, and H. moscheutos. To investigate the monophyly of Hibiscus section Muenchhusia, its relationship to other Hibiscus species, and the phylogenetic relationships among its species, DNA sequence data were obtained. To investigate the placement of section Muenchhusia within Hibiscus, chloroplast ndhF gene and rpL16 intron sequences were generated and added to a recently published dataset. To investigate relationships within section Muenchhusia three DNA sequence data sets were generated: a non-coding region of the chloroplast genome (rpL16 intron), nuclear ribosomal ITS, and a nuclear gene encoding granule-bound starch synthase (GBSSI). Analyses of ndhF + rpL16 data indicated that section Muenchhusia is indeed monophyletic and is embedded in a clade that includes representatives of Hibiscus section Trionum sensu lato plus other genera segregated from Hibiscus (Abelmoschus, Fioria), and the tribe Malvavisceae. Within section Muenchhusia little to no phylogenetically informative variation was detected in the ITS or rpL16 sequences. The GBSSI data, on the other hand, provided sufficient information to resolve relationships among species. The species of section Muenchhusia fall into two primary clades, one consisting of H. grandiflorus and H. moscheutos sensu lato, the other including H. coccineus, H. dasycalyx, and H. laevis. These phylogenetic data corroborate earlier biosystematic studies that also placed the species into these same two groups. Sequence polymorphism was observed in one accession each of H. dasycalyx and H. grandiflorus and resolution of the underlying alleles indicates that gene flow has occurred from H. moscheutos into both H. dasycalyx and H. grandiflorus.

Hybridization between two gartersnake species (Thamnophis) of conservation concern: a threat or an important natural interaction?

Distinguishing between hybrid zones formed by secondary contact versus parapatric divergence-with-gene-flow is an important challenge for understanding the interplay of geographic isolation and local adaptation in the origin of species. Similarly, distinguishing between natural hybrid zones and those that formed as a consequence of recent human activities has important conservation implications. Recent work has demonstrated the existence of a narrow hybrid zone between the plains gartersnake (Thamnophis radix) and Butler’s gartersnake (T. butleri) in the Great Lakes region of North America, raising questions about the history and conservation value of genetically admixed populations. Both taxa are of conservation concern, and it is not clear whether to regard hybridization as a threat or a natural interaction. Here we use phylogeographic and population genetic methods to assess the timescales of divergence and hybridization, and test for evidence that the hybrid zone is of recent origin. We assayed AFLP markers and ND2 mitochondrial DNA (mtDNA) sequences from T. radix, T. butleri, and the closely related short-headed gartersnake (T. brachystoma) throughout their North American ranges. We find shallow mtDNA divergence overall and high levels of variation within the contact zone. These patterns are inconsistent with recent contact of long-diverged taxa. It is not possible to distinguish true divergence-with-gene-flow from a long-term secondary contact zone, but we infer that the hybrid zone is a long-standing, natural interaction.