Polyphyly of the grass tribe Hainardieae (Poaceae: Pooideae): identification of its different lineages based on molecular phylogenetics, including morphological and cytogenetic characteristics

The small pooid grass tribe Hainardieae comprises six genera with approximately ten species; however, this tribe was not accepted by all previous taxonomic treatments. To study the relationships among these genera and to infer the phylogeny and evolutionary patterns, we used sequence variation of the internal transcribed spacers (ITS) of nuclear ribosomal and chloroplast (cp) matK DNA and morphology. Many genera of the Aveneae/Poeae tribe complex additionally were included. Both molecular datasets showed Hainardieae to be highly polyphyletic, and its genera to branch with different groups of the Aveneae/Poeae. Parapholis and Hainardia are corroborated as being closely related, and belonging to a firmly supported Eurasian clade together with Catapodium incl. Scleropoa, Cutandia, Desmazeria, Sphenopus, Vulpiella (subtribe Parapholiinae) and with Cynosurus as sister to this assemblage. The other genera of traditionally recognised Hainardieae are positioned phylogenetically distant: Mediterranean Narduroides is verified as more or less related to Festuca and relatives (subtribe Loliinae), whereas the west Eurasian Pholiurus is close to the lineage of Poa and relatives (subtribe Poinae). North American Scribneria is sister to Deschampsia and both genera should be unified under a common subtribe (Aristaveninae or Holcinae). The phylogenetic position of the Algerian genus Agropyropsis (close to Narduroides and within the Loliinae) is suggested on morphology only, because no molecular data was obtained for it. Considering classification, we support the abandonment of tribe Hainardieae and argue to abandon Poeae subtribe Scribneriinae. Poeae subtribe Parapholiinae is redefined with a novel genus content, due to the exclusion of Agropyropsis and Pholiurus and the inclusion of Vulpiella.     

Phylogenetic origin of Beckmannia （Poaceae） inferred from molecular evidence

The phylogenetic origin of Beckmannia remains unknown. The genus has been placed within the Chlorideae, Aveneae （Agrostideae）, Poeae, or treated as an isolate lineage, Beckmanniinae. In the present study, we used nuclear internal transcribed spacer （ITS） and chloroplast trnL-F sequences to examine the phylogenetic relationship between Beckmannia and those genera that have assumed to be related. On the basis of the results of our studies, the following conclusions could be drawn： （i） Beckmannia and Alopecurus are sister groups with high support; and （ii） Beckmannia and Alopecurus are nested in the Poeae clade with high support. The results of our analysis suggest that Beckmannia should be placed in Poeae.     

基于trnL-F序列探讨菵草植物的系统位置

以淡竹为外类群,用PAUP4.0b10软件采用最大简约法对菵草及其近缘植物的叶绿体trnL-F序列进行了系统发育分析,结果表明:(1)菵草和看麦娘聚成一小支,自展支持率为91%,说明两者之间亲缘关系较近。(2)菵草、看麦娘以及梯牧草与早熟禾族的早熟禾属、碱茅、硬草等聚合为一分支,得到了较高的支持率,而燕麦族的剪股颖属、拂子茅属、野青茅和棒头草组成另一分支,自展支持率为99%,分子证据支持菵草、看麦娘、梯牧草并入早熟禾族。因此,早熟禾族和燕麦族的范围需要重新界定。     

Molecular phylogeny of the Pooideae (Poaceae) based on nuclear rDNA (ITS) sequences

Phylogenetic relationships of the Poaceae subfamily, Pooideae, were estimated from the sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The entire ITS region of 25 species belonging to 19 genera representing seven tribes was directly sequenced from polymerase chain reaction (PCR)-amplified DNA fragments. The published sequence of rice, Oryza saliva, was used as the outgroup. Sequences of these taxa were analyzed with maximum parsimony (PAUP) and the neighbor-joining distance method (NJ). Among the tribes, the Stipeae, Meliceae and Brachypodieae, all with small chromosomes and a basic number more than x=7, diverged in succession. The Poeae, Aveneae, Bromeae and Triticeae, with large chromosomes and a basic number of x=7, form a monophyletic clade. The Poeae and Aveneae are the sister group of the Bromeae and Triticeae. On the ITS tree, the Brachypodieae is distantly related to the Triticeae and Bromeae, which differs from the phylogenies based on restriction-site variation of cpDNA and morphological characters. The phylogenetic relationships of the seven pooid tribes inferred from the ITS sequences are highly concordant with the cytogenetic evidence that the reduction in chromosome number and the increase in chromosome size evolved only once in the pooids and pre-dated the divergence of the Poeae, Aveneae, Bromeae and Triticeae.This paper reports factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitableThis paper is a cooperative investigation of USDA-ARS and the Utah Agricultural Experiment Station. Logan, Utah 84322. Journal Paper No. 4581     

Evolutionary processes in the genus Coreocarpus: insights from molecular phylogenetics

A molecular phylogenetic study of the plant genus Coreocarpus was conducted using nuclear (ITS) and plastid (rpl16 intron) DNA sequences, with phylogenies of the nuclear and plastid sequences highly congruent in defining a monophyletic group of six species (core Coreocarpus), although three other species often placed within the genus were excluded. Relationships within the genus are largely but not totally concordant with prior biosystematic studies. Despite strong molecular support, no morphological characters uniting the six species of core Coreocarpus have been identified; retention of plesiomorphic characters and the genetic lability of characters are two probable factors contributing to lack of consistent defining characters. The age of the core Coreocarpus is estimated at 1 million years because the basal species is endemic to a volcanic island that emerged in the past million years. Mapping the results of earlier breeding studies on the molecular phylogeny showed that use of cross-compatibility as a criterion for species delimitation would result in the recognition of paraphyletic species. Prior field, morphological, and biosystematic studies provided no indication of past hybridization in the evolution of Coreocarpus, and species in the genus appeared to be well defined morphologically. However, three instances of incongruence were observed. Two of these were between the nuclear and plastid partitions, and the third was between the morphological species assignment of one accession and the molecular data. If hybridization accounts for incongruence between the nuclear and plastid data, it occurred between species that now appear to be cross-incompatible and allopatric. The incongruence between morphological species assignment and the molecular data could be the result of parallel fixation of characters that have a simple genetic basis. This study suggests that the evolutionary history of Coreocarpus is much more complex than indicated from prior biosystematic investigations and that biosystematic and molecular phylogenetic studies may complement each other for elucidating the evolution and phylogeny of a group.     

被子植物系统发育深层关系研究: 进展与挑战

被子植物系统发育学是研究被子植物及其各类群间亲缘关系与进化历史的学科。从20世纪90年代起, 核苷酸和氨基酸序列等分子数据开始被广泛运用于被子植物系统发育研究, 经过20多年的发展, 从使用单个或联合少数几个细胞器基因, 到近期应用整个叶绿体基因组来重建被子植物的系统发育关系, 目、科水平上的被子植物系统发育框架已被广泛接受。在这个框架中, 基部类群、主要的5个分支(即真双子叶植物、单子叶植物、木兰类、金粟兰目和金鱼藻目)、每个分支所包含的目以及几个大分支包括的核心类群等都具有高度支持。与此同时, 细胞器基因还存在一些固有的问题, 例如单亲遗传、系统发育信息量有限等, 因此近年来双亲遗传的核基因在被子植物系统发育研究中的重要性逐渐得到关注, 并在不同分类阶元的研究中都取得了一定进展。但是, 被子植物系统发育中仍然存在一些难以确定的关系, 例如被子植物5个分支之间的关系、真双子叶植物内部某些类群的位置等。本文简述了20多年来被子植物系统发育深层关系的主要研究进展, 讨论了被子植物系统发育学常用的细胞器基因和核基因的选用, 已经确定和尚未确定系统发育位置的主要类群, 以及研究中尚存在的问题和可能的解决方法。     

Complex evolution in Arundinarieae (Poaceae: Bambusoideae): incongruence between plastid and nuclear GBSSI gene phylogenies

The monophyly of tribe Arundinarieae (the temperate woody bamboos) has been unequivocally recovered in previous molecular phylogenetic studies. In a recent phylogenetic study, 10 major lineages in Arundinarieae were resolved based on eight non-coding plastid regions, which conflicted significantly with morphological classifications both at the subtribal and generic levels. Nevertheless, relationships among and within the 10 lineages remain unclear. In order to further unravel the evolutionary history of Arundinarieae, we used the nuclear GBSSI gene sequences along with those of eight plastid regions for phylogenetic reconstruction, with an emphasis on Chinese species. The results of the plastid analyses agreed with previous studies, whereas 13 primary clades revealed in the GBSSI phylogeny were better resolved at the generic level than the plastid phylogeny. Our analyses also revealed many inconsistencies between the plastid DNA and the nuclear GBSSI trees. These results implied that the nuclear genome and the plastid genome had different evolutionary trajectories. The patterns of incongruence suggested that lack of informative characters, incomplete lineage sorting, and/or hybridization (introgression) could be the causes. Seven putative hybrid species were hypothesized, four of which are discussed in detail on the basis of topological incongruence, chromosome numbers, morphology, and distribution patterns, and those taxa probably resulted from homoploid hybrid speciation. Overall, our study indicates that the tribe Arundinarieae has undergone a complex evolution.     

On the evolutionary descent of organisms and organelles: a global phylogeny based on a highly conserved structural core in small subunit ribosomal RNA

To probe the earliest evolutionary events attending the origin of the five known genome types (archaebacterial, eubacterial, nuclear, mitochondrial and plastid), we have analyzed sequences corresponding to a ubiquitous, highly conserved core of secondary structure in small subunit rRNA. Our results support (i) the existence of three primary lineages (archaebacterial, eubacterial, and nuclear), (ii) a specific eubacterial ancestry for plastids and mitochondria (plant, animal, fungal), and (iii) an endosymbiotic, evolutionary origin of the two types of organelle from within distinct groups of eubacteria (blue-green algae (cyanobacteria) in the case of plastids, nonphotosynthetic aerobic bacteria in the case of mitochondria). In addition, our analysis suggests (iv) a biphyletic origin of mitochondria, with animal and fungal mitochondria branching together but separately from plant mitochondria, and (v) a monophyletic origin of plastids. The method described here provides a powerful and generally applicable molecular taxonomic approach towards a global phylogeny encompassing all organisms and organelles.     

Phylogenetics and reticulate evolution in Pistacia (Anacardiaceae)

The systematic position and intrageneric relationships of the economically important Pistacia species (Anacardiaceae) are controversial. The phylogeny of Pistacia was assessed using five data sets: sequences of nuclear ribosomal ITS, the third intron of the nuclear nitrate reductase gene (NIA-i3), and the plastid ndhF, trnL-F and trnC-trnD. Significant discordance was detected among ITS, NIA-i3, and the combined plastid DNA data sets. ITS, NIA-i3, and the combined plastid data sets were analyzed separately using Bayesian and parsimony methods. Both the ITS and the NIA-i3 data sets resolved the relationships among Pistacia species well; however, these two data sets had significant discordance. The ITS phylogeny best reflects the evolutionary relationships among Pistacia species. Lineage sorting of the NIA-i3 alleles may explain the conflicts between the NIA-i3 and the ITS data sets. The combined analysis of three plastid DNA data sets resolved Pistacia species into three major clades, within which only a few subclades were supported. Pistacia was shown to be monophyletic in all three analyses. The previous intrageneric classification was largely inconsistent with the molecular data. Some Pistacia species appear not to be genealogical species, and evidence for reticulate evolution is presented. Pistacia saportae was shown to be a hybrid with P. lentiscus (maternal) and P. terebinthus (paternal) as the parental taxa.     

Phylogenetic relationships in the grass family (Poaceae) based on the nuclear single copy locus topoisomerase 6 compared with chloroplast DNA

Phylogenetic relationships within the grass family were studied using a newly obtained locus of the nuclear single copy gene topoisomerase 6 (Topo6) spanning the four exons 8–11 and the chloroplast matK gene. Data were evaluated using maximum parsimony, maximum likelihood and Bayesian methods. All analyses showed genera Streptochaeta and Anomochloa as early diverging, followed by Pharus as sister to the rest of the Poaceae, and monophyly of the subfamily Anomochlooideae was supported by the nuclear dataset. The remaining grasses formed a strongly supported and monophyletic group, which split into the major clades BEP and PACMAD in the Topo6 analyses. Monophyly of the BEP clade was strongly supported by the Topo6 data. The results showed clearly incongruity between the two sets of data, such as the different subfamilial relationships of Bambusoideae, Ehrhartoideae and Pooideae. Most of the analysed species are representatives of subfamily Pooideae, which was analysed in more detail by PCR fragment length differences of another Topo6 region spanning the exons 17–19. Monophyly of Pooideae was strongly supported by the matK data, whereas the nuclear data placed Brachyelytrum outside of the remaining Pooideae. Relationships within the early evolutionary lineages remained largely unresolved in the phylogenetic trees, but the ‘core’ Pooideae (Aveneae/Poeae tribe complex and Hordeeae) were highly supported in all analyses. The differences in amplification lengths illustrate the tribe and subtribe classification of Pooideae. The comparatively conserved structure of the newly studied Topo6 region makes it a promising marker from the nuclear genome that could be successfully PCR-amplified to study higher-level phylogenetic relationships within grasses and perhaps between families within the order Poales.     

Molecular phylogenetic relationships inAveneae (Poaceae) species and other grasses as inferred from ITS1 and ITS2 rDNA sequences

A phylogenetic analysis was conducted on sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA in 23 species ofAveneae (Poaceae subfam.Pooideaae). These sequences ofHelictotrichon spp.,Arrhenatherum elatius, Avena spp.,Trisetum spp.,Koeleria spp.,Holcus lanatus, Alopecurus vaginatus together with published ITS sequences of furtherAveneae, Poeae, Triticeae, andBromeae were analysed by the neighbor-joining distance method to assess the molecular phylogenetic relationship in perennial and annualAveneae. The results suggest unexpectedly close affinities of the agronomically important genusAvena to comparatively small-flowered taxa ofAveneae. GenusArrhenatherum and small-flowered subgenera ofHelictotrichon are close extant relatives. The large genusHelictotrichon is para- if not polyphyletic, only its subgenera are monophyletic.Trisetum is clearly separated fromHelictotrichon and forms together withKoeleria and perhaps others a monophyletic lineage which is characterised by a conspicuous 9-bp deletion in ITS1. The impact of the ITS data on the delineation of some genera and subtribes ofAveneae and on the recognition of their biogeographical and ecological patterns is outlined.     

A phylogenetic analysis of chloroplast DNA restriction site variation inPoaceae subfam.Pooideae

A phylogenetic analysis was conducted on chloroplast DNA restriction site variation in 34 genera of grasses (familyPoaceae), including 28 genera from subfam.Pooideae (representing tribesAveneae, Brachypodieae, Bromeae, Meliceae, Poeae, Stipeae, andTriticeae) and representatives of three other subfamilies,Arundinoideae, Oryzoideae, andPanicoideae. Analyses of all 34 genera always distinguishedPooideae as monophyletic, regardless of which nonpooid genus functioned as outgroup; six separate analyses of all 28 pooid genera, each including one of the six nonpooid genera as outgroup, resolved five identically-constituted clades withinPooideae (in four cases), or (in the other two cases) yielded results that were less well resolved, but not in conflict with those of the other four analyses. The four best-resolved analyses distinguishedMeliceae as the earliest diverging lineage withinPooideae, andStipeae as the next. Above the point of divergence ofStipeae is a dichotomy between supertribeTriticodae (including tribesBrachypodieae, Bromeae, andTriticeae), and a clade comprisingPoeae andAveneae. The analysis supports some tribal realignments, specifically the assignment ofBriza, Chascolytrum, Microbriza, andTorreyochloa toAveneae, andArctagrostis, Catabrosa, andSesleria toPoeae. The analysis also suggests that the pooid spikelet (i.e., glumes shorter than lemmas and florets two or more) is plesiomorphic inPooideae, and that spikelets with one floret, and those with glumes longer than the first lemma, each have evolved more than once withinPooideae. Results also indicate that small chromosomes and chromosome numbers based on x=c. 10–12 are plesiomorphic withinPooideae. Alternative states of these characters (chromosomes large, chromosome numbers based on x=7) are interpreted as synapomorphies or parallelisms of clades that includeTriticodae, Aveneae, andPoeae. Lanceolate lodicule shape may be a synapomorphy of the clade that includesStipeae, Triticodae, Aveneae, andPoeae, and loss of lodicule vascularization a synapomorphy of the entirePooideae.     

Phylogeny reconstruction in the Caesalpinieae grade (Leguminosae) based on duplicated copies of the sucrose synthase gene and plastid markers

The Caesalpinieae grade (Leguminosae) forms a morphologically and ecologically diverse group of mostly tropical tree species with a complex evolutionary history. This grade comprises several distinct lineages, but the exact delimitation of the group relative to subfamily Mimosoideae and other members of subfamily Caesalpinioideae, as well as phylogenetic relationships among the lineages are uncertain. With the aim of better resolving phylogenetic relationships within the Caesalpinieae grade, we investigated the utility of several nuclear markers developed from genomic studies in the Papilionoideae. We cloned and sequenced the low copy nuclear gene sucrose synthase (SUSY) and combined the data with plastid trnL and matK sequences. SUSY has two paralogs in the Caesalpinieae grade and in the Mimosoideae, but occurs as a single copy in all other legumes tested. Bayesian and maximum likelihood phylogenetic analyses suggest the two nuclear markers are congruent with plastid DNA data. The Caesalpinieae grade is divided into four well-supported clades (Cassia, Caesalpinia, Tachigali and Peltophorum clades), a poorly supported clade of Dimorphandra Group genera, and two paraphyletic groups, one with other Dimorphandra Group genera and the other comprising genera previously recognized as the Umtiza clade. A selection analysis of the paralogs, using selection models from PAML, suggests that SUSY genes are subjected to a purifying selection. One of the SUSY paralogs, under slightly stronger positive selection, may be undergoing subfunctionalization. The low copy SUSY gene is useful for phylogeny reconstruction in the Caesalpinieae despite the presence of duplicate copies. This study confirms that the Caesalpinieae grade is an artificial group, and highlights the need for further analyses of lineages at the base of the Mimosoideae.     

PCR AMPLIFICATION OF NUCLEAR AND PLASTID GENES FROM ALGAL HERBARIUM SPECIMENS AND ALGAL SPORES1

Plastid and nuclear ribosomal genes were amplified from an 11-year-old herbarium specimen using simple, rapid, nontoxic, and inexpensive methods. Gonimoblast tissue, isolated from either dried or fresh red algal cystocarps, was ground using the polyvalent, metal chelating resin Chelax 100. After boiling and centrifuging, the supernatant yielding enough DNA for 20 or more polymerase chain reactions. Using these methods, we also amplified plastid and nuclear genes from as few as two red algal spores. These methods should facilitate future studies of algal systematics, evolutionary biogeography, and phylogeny as well as studies of algal dispersal patterns and population biology.     

Reconciling gene and genome duplication events: using multiple nuclear gene families to infer the phylogeny of the aquatic plant family Pontederiaceae

Most plant phylogenetic inference has used DNA sequence data from the plastid genome. This genome represents a single genealogical sample with no recombination among genes, potentially limiting the resolution of evolutionary relationships in some contexts. In contrast, nuclear DNA is inherently more difficult to employ for phylogeny reconstruction because major mutational events in the genome, including polyploidization, gene duplication, and gene extinction can result in homologous gene copies that are difficult to identify as orthologs or paralogs. Gene tree parsimony (GTP) can be used to infer the rooted species tree by fitting gene genealogies to species trees while simultaneously minimizing the estimated number of duplications needed to reconcile conflicts among them. Here, we use GTP for five nuclear gene families and a previously published plastid data set to reconstruct the phylogenetic backbone of the aquatic plant family Pontederiaceae. Plastid-based phylogenetic studies strongly supported extensive paraphyly of Eichhornia (one of the four major genera) but also depicted considerable ambiguity concerning the true root placement for the family. Our results indicate that species trees inferred from the nuclear genes (alone and in combination with the plastid data) are highly congruent with gene trees inferred from plastid data alone. Consideration of optimal and suboptimal gene tree reconciliations place the root of the family at (or near) a branch leading to the rare and locally restricted E. meyeri. We also explore methods to incorporate uncertainty in individual gene trees during reconciliation by considering their individual bootstrap profiles and relate inferred excesses of gene duplication events on individual branches to whole-genome duplication events inferred for the same branches. Our study improves understanding of the phylogenetic history of Pontederiaceae and also demonstrates the utility of GTP for phylogenetic analysis.     

Phylogeny and diversification of Chinese Araliaceae based on nuclear and plastid DNA sequence data

Chinese Araliaceae consist of 20 genera and ca. 175 species. To assess the evolutionary relationships of Araliaceae and their biogeographic diversification in China, the phylogeny of Chinese Araliaceae was constructed by sampling 96 accessions representing 20 genera and 50 species of Chinese Araliaceae and 45 closely related taxa using sequences of the nuclear ribosomal internal transcribed spacer (ITS) region and six plastid regions (the ndhF gene, the trnL-trnF region, the rps16intron, the atpB-rbcL intergenic spacer, the rpl16 intron, and the psbA-trnH intergenic spacer). Phylogenetic analyses of the combined plastid and ITS data supported the results of the previously studies that the Chinese members of Araliaceae were scattered within the Asian Palmate group and the Aralia-Panax group withOsmoxylon at the base of core Araliaceae. The generic status of Pentapanax and Tupidanthus is not supported. Our analysis clearly places them in Aralia and AsianSchefflera, respectively. In a broader phylogenetic framework of Araliaceae, based on the fossil-calibrated Bayesian dating, Chinese Araliaceae was inferred to have originated in Asia and underwent a rapid radiation in its evolutionary history. Its diversification is hypothesized to have been driven largely by the orogenies in Asia during the Cenozoic. In China, the distribution pattern of the phylogenetic diversity of Araliaceae corresponds with its taxonomic diversity across the entire region.     

Highly conserved low-copy nuclear genes as effective markers for phylogenetic analyses in angiosperms

? Organismal phylogeny provides a crucial evolutionary framework for many studies and the angiosperm phylogeny has been greatly improved recently, largely using organellar and rDNA genes. However, low-copy protein-coding nuclear genes have not been widely used on a large scale in spite of the advantages of their biparental inheritance and vast number of choices. ? Here, we identified 1083 highly conserved low-copy nuclear genes by genome comparison. Furthermore, we demonstrated the use of five nuclear genes in 91 angiosperms representing 46 orders (73% of orders) and three gymnosperms as outgroups for a highly resolved phylogeny. ? These nuclear genes are easy to clone and align, and more phylogenetically informative than widely used organellar genes. The angiosperm phylogeny reconstructed using these genes was largely congruent with previous ones mainly inferred from organellar genes. Intriguingly, several new placements were uncovered for some groups, including those among the rosids, the asterids, and between the eudicots and several basal angiosperm groups. ? These conserved universal nuclear genes have several inherent qualities enabling them to be good markers for reconstructing angiosperm phylogeny, even eukaryotic relationships, further providing new insights into the evolutionary history of angiosperms.     

Quality and quantity of data recovered from massively parallel sequencing: Examples in Asparagales and Poaceae

? Premise of the study: Genome survey sequences (GSS) from massively parallel sequencing have potential to provide large, cost-effective data sets for phylogenetic inference, replace single gene or spacer regions as DNA barcodes, and provide a plethora of data for other comparative molecular evolution studies. Here we report on the application of this method to estimating the molecular phylogeny of core Asparagales, investigating plastid gene losses, assembling complete plastid genomes, and determining the type and quality of assembled genomic data attainable from Illumina 80-120-bp reads. ? Methods: We sequenced total genomic DNA from samples in two lineages of monocotyledonous plants, Poaceae and Asparagales, on the Illumina platform in a multiplex arrangement. We compared reference-based assemblies to de novo contigs, evaluated consistency of assemblies resulting from use of various references sequences, and assessed our methods to obtain sequence assemblies in nonmodel taxa. ? Key results: Our method returned reliable, robust organellar and nrDNA sequences in a variety of plant lineages. High quality assemblies are not dependent on genome size, amount of plastid present in the total genomic DNA template, or relatedness of available reference sequences for assembly. Phylogenetic results revealed familial and subfamilial relationships within Asparagales with high bootstrap support, although placement of the monotypic genus Aphyllanthes was placed with moderate confidence. ? Conclusions: The well-supported molecular phylogeny provides evidence for delineation of subfamilies within core Asparagales. With advances in technology and bioinformatics tools, the use of massively parallel sequencing will continue to become easier and more affordable for phylogenomic and molecular evolutionary biology investigations.