Apomixis and reticulate evolution in the Asplenium monanthes fern complex

Abstract

Background and aims: Asexual reproduction is a prominent evolutionary process within land plant lineages and especially in ferns. Up to 10 % of the approx. 10 000 fern species are assumed to be obligate asexuals. In the Asplenium monanthes species complex, previous studies identified two triploid, apomictic species. The purpose of this study was to elucidate the phylogenetic relationships in the A. monanthes complex and to investigate the occurrence and evolution of apomixis within this group.

Methods: DNA sequences of three plastid markers and one nuclear single copy gene were used for phylogenetic analyses. Reproductive modes were assessed by examining gametophytic and sporophyte development, while polyploidy was inferred from spore measurements.

Key results: Asplenium monanthes and A. resiliens are confirmed to be apomictic. Asplenium palmeri, A. hallbergii and specimens that are morphologically similar to A. heterochroum are also found to be apomictic. Apomixis is confined to two main clades of taxa related to A. monanthes and A. resiliens, respectively, and is associated with reticulate evolution. Two apomictic A. monanthes lineages, and two putative diploid sexual progenitor species are identified in the A. monanthes clade.

Conclusions: Multiple origins of apomixis are inferred, in both alloploid and autoploid forms, within the A. resiliens and A. monanthes clades.

Fig. 1.

Sexual vs. apogamous prothalli. Pictures of cultivated prothalli from different specimens (Supplementary Data, Table S7): (A) sexual A. formosum, RD28 – the dashed arrow show the archegonia; (B) apogamous A. monanthes, RD110; (C) apogamous A. monanthes, RD101b; (D) apogamous A. resiliens, RD127; (E) apogamous A. resiliens, RD128; (F) apogamous A. monanthes, RD132; (G) apogamous A. resiliens RD107; (H) apogamous A. monanthes, RD17. Filled circles indicate apical meristems, and sporophytes are indicated by filled squares. Solid arrows indicate apogamous growth.

Fig. 2.

Bayesian phylogenetic tree based on the combined plastid data. Posterior support values of P > 0·6 are shown. Tips are labelled with species names followed by a voucher accession, and colour-coded according to species. Sub-clades are abbreviated and summarized to the right of the tree. Abbreviations for sub-clades correspond to those used in the text. Asterisks indicate tips representing multiple haplotypes (Supplementary Data, Table S3). Filled black circles indicate apomictic specimens, and open circles indicate specimens inferred to have a sexual mode of reproduction.

Fig. 3.

Bayesian phylogenetic tree based on the nuclear data. Posterior support values of P > 0·6 are shown. Tips are labelled with species names, followed by a voucher accession (e.g. RD112), and indication of the clone identifier out of the total number of clones (e.g. C2/3 = clone no. 2 out of three unique clones; see text for details). Tips are colour-coded according to species. Sub-clades are abbreviated and summarized to the right of the tree.

Fig. 4.

A reticulation network illustrating the evolutionary history of the A. monanthes complex. This summarizes a hybridization network (Supplementary Data, Fig. S6) performed in SplitsTree (Huson and Bryant, 2006) and illustrates hypothetical hybrid relationships observable by comparison between nuclear and plastid trees (Figs 1 and 2). The terminals illustrated are inferred as different species forms. Where copy numbers for one-species forms are variable, we have included the range of values in parentheses after the species label. Branches are colour-coded according to species. Filled black circles indicate apomictic species, and open circles indicate species inferred to have a sexual mode of reproduction. The plastid group and nuclear copy distribution are summarized adjacent to terminal labels. The dashed red line indicates inferred association of this spec.nov.1 with the MO1 A. monanthes lineage.

Fig. 5.

Boxplots illustrating variation in spore length of species/specimens of the A. monanthes complex. Each boxplot is labelled below with its corresponding species name and specimen voucher, and coloured according to species as in Fig. 2. Boxplots are grouped together according to clades and ordered from lowest to highest mean lengths. Each boxplot represents the variance of measurements of spores within each specimen, the thick horizontal line is the median, the box indicates the variation observed between the 25th and 75th percentiles, the whiskers show the variance range, and small circles identify extreme outliers. Dashed lines running across the graph illustrate average spore measurements in comparison with the ploidy levels of the spores, based on average counts of triploid A. resiliens (spore ploidy = 3x) specimens, hexaploid A. heterochroum (spore ploidy = 3x) specimens (Morzenti, 1966) and diploids and tetraploids of the A. trichomanes complex (spore ploidy = x and 2x, respectively) (Tutin et al., 1993). Filled black circles indicate apomictic specimens that produce 32 spores per sporangium. Open circles indicate specimens that produce 64 spores per sporangium and are inferred to have a sexual mode of reproduction. The asterisk indicates a specimen that produces both 32 and 64 spores per sporangium.

Fig. 6.

A reticulogram illustrating the apomictic polyploid species relationships and hypothetical origins of apomixis within the A. monanthes clade. Extant cytotypes are used to illustrate hypothesized lineage origins and parental relationships. The inferred ploidy levels within the three lineages are represented using circles for diploids, triangles for triploids and squares for tetraploids. The diploids are sexually reproducing species, whereas the triploids and tetraploid forms are apomictic. The dashed circle represents a hypothesized (either unsampled or extinct) parent cytotype. Dashed arrows indicate paternal parent relationships. Solid arrows indicate maternal parents (i.e. unreduced gametophytes).