Phylogeny of Dorstenia (Moraceae) reveals the polyphyletic nature of its neotropical sections

Marcelo Dias Machado Filho Leandro Cardoso Pederneiras Vitor Hugo Maia Andrea Ferreira da Costa Vidal de Freitas Mansano About the authors

Abstract

Dorstenia, the second largest genus of Moraceae, comprises nine sections that are mainly found in Africa and America. Two of them are woody macrospermous, and the other seven are herbaceous microspermous. There are three sections in the Neotropics, all of which are herbaceous and taxonomically complex owing to their great morphological similarity. The most recent molecular phylogenetic studies of Dorstenia suggested that the neotropical sections are polyphyletic. These studies also showed that the neotropical species represent a sister group to an African woody macrospermous grade rather than African herbaceous microspermous plants. We have now expanded the number of taxa sampled and included other molecular markers to determine whether the previous phylogeny are to be corroborated or whether new taxonomic interpretations are to be followed. This study inferred the phylogeny of the group based on ITS, ETS, and trnL-F regions from 40 of the 58 neotropical species and added a new African taxon, thus including 17 of the 60 known species. Our results reaffirmed the polyphyletic nature of the neotropical sections. Dorstenia sect. Acauloma emerged within the main clade of D. sect. Kosaria (both African species), a result that confirms the affinity of these taxa already observed in previous morphological studies. We suggest Dorstenia sect. Dorstenia as the only neotropical section.

Key words
molecular markers; neotropics; phylogeny; plant evolution; rosids

Resumo

Dorstenia, o segundo maior gênero de Moraceae, compreende nove seções encontradas principalmente na África e na América. Duas delas são macrospermas lenhosas e os outros sete são microspermas herbáceas. Existem três seções na região Neotropical, todas herbáceas e taxonomicamente complexas devido à sua grande semelhança morfológica. Os estudos filogenéticos moleculares mais recentes sugerem que as seções neotropicais são polifiléticas. Esses estudos também mostraram que as espécies neotropicais representam um grupo irmão de um grado de macrospermas lenhosos africanos, em vez de microspermas herbáceos. No presente trabalho, expandimos o número de taxa amostrados e incluímos outros marcadores moleculares para determinar se a filogenia anterior deve ser corroborada e se novas interpretações taxonômicas devem ser seguidas. Este estudo inferiu a filogenia do grupo com base nas regiões ITS, ETS e trnL-F de 40 das 58 espécies neotropicais e adicionou um novo táxon africano, incluindo 17 das 60 espécies conhecidas. Nossos resultados reafirmaram a natureza polifilética das seções neotropicais. Dorstenia sect. Acauloma surgiu dentro do clado principal de D. sect. Kosaria (ambas espécies africanas), resultado que confirma a afinidade desses táxons já observada em estudos morfológicos anteriores. Nós sugerimos Dorstenia sect. Dorstenia com a única seção neotropical, porém maiores estudos moleculares são necessários.

Palavras-chave
marcadores moleculares; Neotrópico; filogenia; evolução das plantas; Rosídeas

Introduction

The mulberry family, Moraceae, contains 39 genera and approximately 1,100 species that are globally distributed throughout tropical and temperate regions (Berg 2001Berg CC (2001) Moreae, Artocarpeae and Dorstenia (Moraceae): with introductions to the family and Ficus and with additions and corrections to Flora Neotropica 7. Flora Neotropica vol. 83. New York Botanical Garden, New York. 346p.; Zerega & Gardner 2019Zerega NJC & Gardner EM (2019) Delimitation of the new tribe Parartocarpeae (Moraceae) is supported by a 333-gene phylogeny and resolves tribal level Moraceae taxonomy. Phytotaxa 388: 253-265. ). Dorstenia L. is the second largest genus of Moraceae, with approximately 115 species (Berg & Hijman 1999Berg CC & Hijman MEE (1999) The genus Dorstenia (Moraceae). Ilicifolia 2: 1-211.; Vianna-Filho et al. 2016Vianna-Filho MDM, Maia VH, Mansano VF & Costa AF (2016) Hijmania, a replacement name for Maria (Moraceae). Phytotaxa 247: 97-98.). Clement & Weiblen (2009)Clement WL & Weiblen GD (2009) Morphological evolution in the mulberry family (Moraceae). Systematic Botany 34: 530-552., Gardner et al. (2017)Gardner EM, Sarraf P, Williams EW & Zerega NJC (2017) Phylogeny and biogeography of Maclura (Moraceae) and the origin of an anachronistic fruit. Molecular Phylogenetics and Evolution 117: 49-59., and Zerega & Gardner (2019)Zerega NJC & Gardner EM (2019) Delimitation of the new tribe Parartocarpeae (Moraceae) is supported by a 333-gene phylogeny and resolves tribal level Moraceae taxonomy. Phytotaxa 388: 253-265. suggested that this genus together with Brosimum, Trymatococcus, Helianthostylis and others comprise the tribe Dorstenieae. The species are distributed in tropical America (ca. 45 species), Africa (ca. 60 species), India, and Sri Lanka (1 species; Carauta 1978Carauta JPP (1978) Dorstenia L. (Moraceae) do Brasil e países limítrofes. Rodriguésia 29: 71-73.; Berg & Hijman 1999Berg CC & Hijman MEE (1999) The genus Dorstenia (Moraceae). Ilicifolia 2: 1-211.; Berg 2001Berg CC (2001) Moreae, Artocarpeae and Dorstenia (Moraceae): with introductions to the family and Ficus and with additions and corrections to Flora Neotropica 7. Flora Neotropica vol. 83. New York Botanical Garden, New York. 346p.) where they inhabit the undergrowth of lowland rainforests, slopes near streams, and rocky areas (Carauta 1978Carauta JPP (1978) Dorstenia L. (Moraceae) do Brasil e países limítrofes. Rodriguésia 29: 71-73.; Berg 2001Berg CC (2001) Moreae, Artocarpeae and Dorstenia (Moraceae): with introductions to the family and Ficus and with additions and corrections to Flora Neotropica 7. Flora Neotropica vol. 83. New York Botanical Garden, New York. 346p.).

The genus Dorstenia is morphologically characterized by its herbaceous habit, but there are a few succulent and woody species (Fig. 1). Their inflorescences are primarily monoecious, with a flattened, expanded receptacle (the coenanthium) with minute both staminate and pistillate flowers that are often tightly packed together; drupaceous fruits with an explosive dehiscence; and seeds that are either macrospermous or microspermous (Berg 2001Berg CC (2001) Moreae, Artocarpeae and Dorstenia (Moraceae): with introductions to the family and Ficus and with additions and corrections to Flora Neotropica 7. Flora Neotropica vol. 83. New York Botanical Garden, New York. 346p.; De Granville 1971De Granville JJ (1971) Notes sur la biologie florale de quelques espèces du genre Dorstenia (Moracées). Cahiers ORSTOM, Série Biologie 15: 61-97.; Misiewicz & Zerega 2012Misiewicz T & Zerega NJC (2012) Phylogeny, biogeography and character evolution of Dorstenia (Moraceae). Edinburgh Journal of Botany 69: 413-440.; Vianna-Filho et al. 2016Vianna-Filho MDM, Maia VH, Mansano VF & Costa AF (2016) Hijmania, a replacement name for Maria (Moraceae). Phytotaxa 247: 97-98.).

Figure 1
a-e. Examples of Dorstenia’s diversity – a-d. Neotropical species – a. D. elata, a well-known D. sect. Lecania with long stems; b. D. cayapia, a geophyte species within D. sect. Emygdioa; c-d. D. arifolia and D. ramosa, respectively, two closely related species within D. sect. Dorstenia, which differ in their inflorescence; e. D. foetida, a Paleotropical succulent species of D. sect. Kosaria.

Berg & Hijman (1999)Berg CC & Hijman MEE (1999) The genus Dorstenia (Moraceae). Ilicifolia 2: 1-211. and Berg (2001)Berg CC (2001) Moreae, Artocarpeae and Dorstenia (Moraceae): with introductions to the family and Ficus and with additions and corrections to Flora Neotropica 7. Flora Neotropica vol. 83. New York Botanical Garden, New York. 346p. subdivided the genus into nine sections: Dorstenia sect. Nothodorstenia Engl. (five woody species in Africa), D. sect. Xylodorstenia Hijman (six woody species in Africa), D. sect. Lecania Carauta (ca. 25 herbaceous species in the Neotropics and two in Africa), D. sect. Lomatophora Hijman (26 herbaceous species in Africa), D. sect. Dorstenia (12 herbaceous species in the Neotropics, sensu Carauta 1978Carauta JPP (1978) Dorstenia L. (Moraceae) do Brasil e países limítrofes. Rodriguésia 29: 71-73.), D. sect. Kosaria (Forssk.) Fisch. & C.A. Mey. (ca. 20 caulescent species in Africa and Asia), D. sect. Bazzemia Hijman (one acaulescent species in Mozambique), D. sect. Emygdioa Carauta (ca. 20 acaulescent species in the Neotropics), and D. sect. Acauloma Hijman (three succulent acaulescent tubiferous species in Africa). Carauta (1978)Carauta JPP (1978) Dorstenia L. (Moraceae) do Brasil e países limítrofes. Rodriguésia 29: 71-73., Berg (2001)Berg CC (2001) Moreae, Artocarpeae and Dorstenia (Moraceae): with introductions to the family and Ficus and with additions and corrections to Flora Neotropica 7. Flora Neotropica vol. 83. New York Botanical Garden, New York. 346p., Vianna-Filho (2012)Vianna-Filho MDM (2012) Filogenia de Dorstenia sect. Dorstenia e revisão taxonômica do clado arifolia. Tese de doutorado, Museu Nacional/Universidade Federal do Rio de Janeiro, Rio de Janeiro. 186p., and Santos et al. (2016)Santos A, José PAS, Vianna Filho MDM & Romaniu-Neto S (2016) Dorstenia (Moraceae) da região da Serra da Mantiqueira, Brasil. Rodriguésia 67: 237-250. pointed out that the circumscription of the three neotropical sections is problematic (D. sect. Dorstenia, D. sect. Emygdioa, and D. sect. Lecania) because there are no clear morphological character states to segregate them.

Although the last taxonomic revisions (Berg & Hijman 1999Berg CC & Hijman MEE (1999) The genus Dorstenia (Moraceae). Ilicifolia 2: 1-211.; Berg 2001Berg CC (2001) Moreae, Artocarpeae and Dorstenia (Moraceae): with introductions to the family and Ficus and with additions and corrections to Flora Neotropica 7. Flora Neotropica vol. 83. New York Botanical Garden, New York. 346p.) have proposed a sectional classification and discussed the closest phylogenetic relationships between these groups and species, there are few studies based on the molecular phylogeny of Dorstenia. The two best-sampled phylogenetic analyses of Dorstenia (Misiewicz & Zerega 2012Misiewicz T & Zerega NJC (2012) Phylogeny, biogeography and character evolution of Dorstenia (Moraceae). Edinburgh Journal of Botany 69: 413-440.; Zhang et al. 2019Zhang Q, Gardner E, Zerega N & Sauquet H (2019) Long-distance dispersal shaped the diversity of tribe Dorstenieae (Moraceae). BioRxiv 531855. doi: <http://dx.doi.org/10.1101/531855>) considered the genus to be monophyletic and suggested that the previous taxonomical sections were polyphyletic. An interesting finding was the position of D. elliptica Bureau, a woody macrospermous plant, as a sister of all neotropical species (Misiewicz & Zerega 2012Misiewicz T & Zerega NJC (2012) Phylogeny, biogeography and character evolution of Dorstenia (Moraceae). Edinburgh Journal of Botany 69: 413-440.; Zhang et al. 2019Zhang Q, Gardner E, Zerega N & Sauquet H (2019) Long-distance dispersal shaped the diversity of tribe Dorstenieae (Moraceae). BioRxiv 531855. doi: <http://dx.doi.org/10.1101/531855>).

New possibilities have emerged after fundamental analyses by two molecular studies on Dorstenia, which mainly focused on biogeography and character evolution (Misiewicz & Zerega 2012Misiewicz T & Zerega NJC (2012) Phylogeny, biogeography and character evolution of Dorstenia (Moraceae). Edinburgh Journal of Botany 69: 413-440.; Zhang et al. 2019Zhang Q, Gardner E, Zerega N & Sauquet H (2019) Long-distance dispersal shaped the diversity of tribe Dorstenieae (Moraceae). BioRxiv 531855. doi: <http://dx.doi.org/10.1101/531855>). In the Neotropics, for example, it was necessary to sample a larger number of taxa in order to determine the degree of monophyletic or polyphyletic sections/species evidenced by the first phylogenies. Misiewicz & Zerega (2012)Misiewicz T & Zerega NJC (2012) Phylogeny, biogeography and character evolution of Dorstenia (Moraceae). Edinburgh Journal of Botany 69: 413-440. analyzed 15 neotropical (27% of the total) and 18 African (30% of the total) species, and Zhang et al. (2019)Zhang Q, Gardner E, Zerega N & Sauquet H (2019) Long-distance dispersal shaped the diversity of tribe Dorstenieae (Moraceae). BioRxiv 531855. doi: <http://dx.doi.org/10.1101/531855> analyzed 25 neotropical (55% of the total), 29 African (48% of the total), and one Asian (100% of the total) species. By increasing the set of neotropical taxa analyzed and using three molecular markers for the analysis, we can improve our understanding on the neotropical evolutionary history and make better decisions about the taxonomic subdivisions (sections).

Our objectives were to determine whether the genus Dorstenia is monophyletic and if the sections remain polyphyletic after with the increase of taxa sampled in the phylogeny. We also discuss the phylogenetic groupings based on morphological data, and propose further taxonomic changes.

Material & Methods

Taxon sampling

Our sampling included 57 species of ca. 115 Dorstenia species currently recognized (e.g., Carauta 1978Carauta JPP (1978) Dorstenia L. (Moraceae) do Brasil e países limítrofes. Rodriguésia 29: 71-73.; Berg & Hijman 1999Berg CC & Hijman MEE (1999) The genus Dorstenia (Moraceae). Ilicifolia 2: 1-211.; Berg 2001Berg CC (2001) Moreae, Artocarpeae and Dorstenia (Moraceae): with introductions to the family and Ficus and with additions and corrections to Flora Neotropica 7. Flora Neotropica vol. 83. New York Botanical Garden, New York. 346p.; Castro & Rappini 2010Castro RM & Rappini A (2010) Flora da Bahia - Moraceae. Sitientibus ser. Ciências Biológicas 10: 97-137.; McCoy & Massara 2008McCoy TA & Massara M (2008) Dorstenia lavrani McCoy & Massara (family Moraceae). Cactus and Succulent Journal 80: 79.; Santos & Romaniuc 2012Santos A & Romaniuc-Neto S (2012) A new species of Dorstenia (Moraceae) from southeastern Brazil. Phytokeys 12: 47-51.; Santos et al. 2013; Machado & Vianna-Filho 2012Machado AFP & Vianna-Filho MDM (2012) Dorstenia romaniucii (Moraceae), a new species from the Brazilian Atlantic Rain Forest. Systematic Botany 37: 451-455.; Chase et al. 2013Chase MW, Thijs KW, Kamau P & Fay MF (2013) Dorstenia christenhuszii (Moraceae), a new species from the Taita Hills, Kenya. Phytotaxa 81: 45-48.; Leal 2014Leal ME (2014) Dorstenia luamensis (Moraceae), a new species from eastern Democratic Republic of Congo. PhytoKeys 42: 49-55.; Machado et al. 2014Machado AFP, Pereira JF & Carauta JPP (2014). Dorstenia triseriata (Moraceae) a new and endangered species from Brazil. PhytoKeys 38: 31-35.; Rzepecky 2016Rzepecky A (2016) Dorstenia horwoodii Rzepecky sp. nov. from Nudum to Novum, a Fortyish Year Hiatus. Cactus and Succulent Journal 88: 66-74.), encompassing 72% of the neotropical species (40 species out of ca. 55) and 28% (17 species out of ca. 60) of the African species. In some cases, two or more individuals per species were analyzed, a total of 92 terminals (88 Dorstenia plus four outgroup terminals; see Table 1 for a list of sequences and their provenance). The phylogeny was rooted in Helianthostylis sprucei Baill., Brosimum guianense (Aubl.) Huber, B. alicastrum Sw., and Trymatococcus amazonicus Poepp. & Endl.

Table 1
Voucher information and Genbank accession numbers of the plant material included in this study. GenBank codes in bold are the new molecular sequences generated by this article. The number next to the species name is the laboratory number.

The relationships among neotropical Dorstenia species were analyzed by sampling taxa from different sections, including samples from the type localities. Dorstenia sect. Dorstenia was represented by 14 species (including the type species), D. sect. Lecania was represented by 14 species out of ca. 25 species (including the type species), and D. sect. sect. Emygdioa was represented by 12 out of 20 species (including the type species).

We did not use the HQ214090, HQ214096, and HQ214105 samples, which represent, according to Misiewicz & Zerega (2012)Misiewicz T & Zerega NJC (2012) Phylogeny, biogeography and character evolution of Dorstenia (Moraceae). Edinburgh Journal of Botany 69: 413-440., the African taxa Dorstenia variifolia Engl., D. tayloriana Rend., and D. cuspidata Hochst. ex A. Rich., respectively, despite the fact that they seem to have emerged from within the neotropical clade. However, this was a very doubtful result due to the strong morphological dissimilarity between the African and neotropical species. The study by Zhang et al. (2019)Zhang Q, Gardner E, Zerega N & Sauquet H (2019) Long-distance dispersal shaped the diversity of tribe Dorstenieae (Moraceae). BioRxiv 531855. doi: <http://dx.doi.org/10.1101/531855> confirmed that these taxa were not part of the neotropical clade and, therefore, we have not included them in our data matrix.

DNA extraction and sequencing

Genomic DNA was extracted from leaf material that had been dried in silica gel according to the CTAB protocol of Doyle & Doyle (1987)Doyle JJ & Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry 19: 11-15.. The plant material was ground in Eppendorf tubes containing metal beads, and the DNA samples were stored in the Brazilian Flora DNA Bank of the Rio de Janeiro Botanical Garden.

The phylogenetic relationships among the neotropical species were inferred using three molecular markers: ITS4/ITS5 for the internal transcribed spacer region (ITS; White et al. 1990White TJ, Bruns T, Lee S & Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ & White TJ (eds.) PCR Protocols: a guide to methods and applications. Academic Press, New York. Pp. 315-322.) of nuclear ribosomal DNA, the trnL-F region (including the trnL intron and the trnL-F spacer; Taberlet et al. 1991Taberlet P, Gielly L, Pautou G & Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105-1109.), and the external transcribed spacer regions (ETS-Hel-1/18S-ETS; Baldwin & Markos 1998Baldwin BG & Markos S (1998) Phylogenetic utility of the external transcribed spacer (ETS) of 18S-26S rDNA: congruence of ETS and ITS trees of Calycadenia (Compositae). Molecular Phylogenetics and Evolution 10: 449-463.) (Tab. 2). These regions can be used to resolve relationships among and/or within the Moraceae genera (e.g., Weiblen 2000Weiblen GD (2000) Phylogenetic relationships of functionally dioecious Ficus (Moraceae) based on ribosomal DNA sequences and morphology. American Journal of Botany 87: 1342-1357.; Datwyler & Weiblen 2004Datwyler SL & Weiblen GD (2004) On the origin of the fig: phylogenetic relationships of Moraceae from ndhF sequences. American Journal of Botany 91: 767-77.; Weiguo et al. 2005Weiguo Z, Yile P, Shihai ZZJ, Xuexia M & Yongping H (2005) Phylogeny of the genus Morus (Urticales: Moraceae) inferred from ITS and trnL-F sequences. African Journal of Biotechnology 4: 563-569.; Rønsted et al. 2008; Clement & Weiblen 2009Clement WL & Weiblen GD (2009) Morphological evolution in the mulberry family (Moraceae). Systematic Botany 34: 530-552.; Pederneiras et al. 2015Pederneiras LC, Romaniuc-Neto S & Mansano VF (2015) Molecular phylogenetics of Ficus sect. Pharmacosycea and the description of Ficus subsect. Carautaea (Moraceae). Systematic Botany 40: 504-509.).

Table 2
Primers and DNA regions analyzed in the present study.

The polymerase chain reaction (PCR) preparations contained 25 ng of DNA template, 1 × reaction buffer (10 × 10 mM Tris-HCl, pH 8.5, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin), 0.2 mM dNTPs, 10 pmol of each primer, and 2.5 units of Taq DNA polymerase, which resulted in a final volume of 50 µL. The ITS and ETS primers made up 4% of the DMSO total reaction volume. The BSA was added to a final concentration of 0.5 µg/µL. The following PCR profiles were used: trnL-F: 94 °C for 2 min, 35 cycles: 94 °C for 1 min, 48 °C for 1 min, 72 °C for 1 min, and 72 °C for 7 min; ITS: 94 °C for 5 min, 30 cycles: 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, and 72 °C for 7 min; ETS: 94 °C for 1 min, 40 cycles: 94 °C for 30 sec, 55 °C for 30 sec, 72 °C for 30 min, and 72 °C for 5 min.

The PCR products were purified and sequenced at Macrogen Inc., Seoul, South Korea. The sequencing was conducted under the BigDyeTM Terminator v3.1 cycling conditions. Then, the PCR products were purified using ethanol precipitation and run using an ABI3730XL automatic sequencer. All sequences generated for this study were deposited in GenBank (Tab. 1).

The sequences were assembled and edited with Geneious Pro 5.0.4 software (Biomatters Ltd.). Prior to assembly, the sequences were trimmed based on the quality values of the traces using the Modified-Mott algorithm, which is part of the software. The contig quality was assessed using the confidence mean value, which is the mean of the confidence scores for the contig base calls. Sequence alignments were conducted by Muscle 3.7 (Edgar 2004Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792-1797.) using the default parameters, and subsequently checked by visual inspection.

Phylogenetic analyses

Our study included two phylogenetic approaches (maximum likelihood [ML] and Bayesian inference [BI]) for each of the four datasets (ETS, ITS, trnL-F, and all together), and there was total of eight phylogenetic trees (strict consensus tree in ML; 50% majority consensus in BI). The best-fit model was estimated for nucleotide substitution by AIC, which is part of the jModeltest (version 0.0.1) package, and the model selected was GTR+G. In the ML analysis, RAxML 8.2.12 (Stamatakis 2014Stamatakis A (2014) RAxML Version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313.) was used for phylogeny estimation with the default settings, and the data partitioned by the alignment region. The BI analysis was performed using MrBayes 3.2.7 (Ronquist et al. 2012Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA & Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539-542.). Two separate runs of four concurrent runs (one cold and three heated) over 30,000,000 generations were employed with sampling at every 3,000 generations. The sampled trees were summarized and those saved prior to the stationarity of the likelihood (burn-in) were excluded. CIPRES Science Gateway (Miller et al. 2010) was used for the ML and BI analyses. The ASTRAL (Mirarab et al. 2014Mirarab S, Reaz R, Bayzid MS, Zimmermann T, Swenson MS & Warnow T (2014) ASTRAL: genome-scale coalescent-based species tree estimation. Bioinformatics 30: i541-i548.) package was used to perform an ML analysis of the three independent molecular markers and to analyze the disagreement between the regions.

Results

This study generated 133 new DNA sequences: 52 from ITS, 43 from trnL-F, and 38 from ETS. The analysis with the ITS marker produced the same groups as the ETS and trnL-F analyses, but there was a larger set of species (92 terminals). The combined analysis of the markers (ETS, ITS, and trnL-F) produced a tree that was similar to that obtained using the ITS marker alone, but with better resolution in some terminal branches (e.g., Dorstenia carautae C.C. Berg and D. milaneziana Carauta, C. Valente & Sucre). The proportion of input quartet trees satisfied by the final ASTRAL species tree was 0.98. According to Mirarab et al. (2014)Mirarab S, Reaz R, Bayzid MS, Zimmermann T, Swenson MS & Warnow T (2014) ASTRAL: genome-scale coalescent-based species tree estimation. Bioinformatics 30: i541-i548., the higher this value, the less disagreement the gene trees have. On this basis, we opted to discuss the phylogenetic hypothesis based on the combined analysis of the markers. We chose to use ML out of the two analyses (ML and BI) because there were no polytomic branches (Fig. 2 and Appendix S1, available on supplementary material <https://doi.org/10.6084/m9.figshare.16569552.v1>).

Figure 2
Maximum Likelihood tree for combined molecular data (ETS + ITS + trnL-F) of 57 Dorstenia species (+ four outgroup species), representing eight sections. The length of the branches represents the genetic distance of each taxon. Pa = Paleotropical species; Ne = Neotropical species; SA = South American species; CA = Central American species. Branches in red indicate posterior probability < 0.9 or bootstrap < 90% (from the Bayesian Inference analysis, appendix). ** = a single woody macrospermous plant within the clade of herbaceous microspermous plants.

Among the paleotropical taxa, the Dorstenia sect. Xylodorstenia (BS 100%, PP 1.0) formed a monophyletic group and D. sect. Nothodorstenia formed a polyphyletic group with a solitary branch (D. elliptica) sister to all neotropical species, but this was inconclusive because of low support (BS 50%, PP < 0.5). Dorstenia sect. Kosaria formed a paraphyletic group because it included D. barnimiana Schweinf. (D. sect. Acauloma; BS 100%, PP 1.0). Dorstenia sect. Lomatophora formed a monophyletic group sister to D. sect. Kosaria and D. sect. Acauloma with moderate to high support (BS 79%; PP 1.0).

The three groups among the predominantly neotropical sections (Dorstenia sect. Dorstenia, D. sect. Lecania, and D. sect. Emygdioa) were polyphyletic with high support (BS = 100%, PP = 1.0) in most of the deep branches. Only D. picta Bureau (one of the two African species of D. sect. Lecania) emerged, along with the paleotropical species, next to the main clade of D. sect. Kosaria and D. sect. Acauloma, but had low support (BS 27%; PP 0.56). Within the predominantly neotropical clade, two branches emerged from the deepest node of the tree. One contained the South American species (BS 44%, PP 0.93) and the other contained the Central American species (BS 98%, PP 1.0).

Discussion

The analysis undertaken in this study suggested that Dorstenia forms a monophyletic group with both bootstrap and posterior probability values higher than 90% for the first and 0.9 for the second one. According to studies by Carauta (1978)Carauta JPP (1978) Dorstenia L. (Moraceae) do Brasil e países limítrofes. Rodriguésia 29: 71-73. and Berg & Hijman (1999)Berg CC & Hijman MEE (1999) The genus Dorstenia (Moraceae). Ilicifolia 2: 1-211., Dorstenia differs from other genera of Moraceae because it mainly consists of herbaceous plants (except for 11 African species), with bisexual inflorescences (except Dorstenia cayapia Vell.) that are discoid (saucer-shaped to cup-shaped), with pistillate flowers immersed in the receptacle, and bracts on the outer surface. All these synapomorphies are key evolutionary traits in the evolution of the group (Misiewicz & Zerega 2012Misiewicz T & Zerega NJC (2012) Phylogeny, biogeography and character evolution of Dorstenia (Moraceae). Edinburgh Journal of Botany 69: 413-440.) that support and identify the species within this clade.

Almost all paleotropical woody macrospermous plants emerged on the deepest nodes of the Dorstenia phylogenetic tree. Dorstenia sect. Xylodorstenia formed a monophyletic group, and D. sect. Nothodorstenia was a polyphyletic group. This early divergent position was expected because almost 90% of the Moraceae species are woody plants (only Dorstenia and Fatoua Gaudich. contain herbaceous species). The polyphyly of D. sect. Nothodorstenia section is an intriguing result. This section, together with D. sect. Xylodorstenia, only contains woody macrospermous plants. However, according to our results, D. elliptica emerges within the set of herbaceous microspermous plants.

The results suggested the validity of the hypothesis that Dorstenia sect. Nothodorstenia is phylogenetically closer to the neotropical clade (D. sect. Dorstenia, D. sect. Emygdioa, and D. sect. Lecania) rather than to the paleotropical sections. Berg & Hijman (1999)Berg CC & Hijman MEE (1999) The genus Dorstenia (Moraceae). Ilicifolia 2: 1-211. mentioned that retainment of bracts (Dorstenia sect. Nothodorstenia, D. sect. Dorstenia, D. sect. Emygdioa, and D. sect. Lecania) indicated the existence of a more recent common ancestor for these two groups compared to any other group, including D. sect. Xylodorstenia. The results indicated that this theory appears to be at least partially correct since D. elliptica (D. sect. Nothodorstenia) may be sister to all neotropical plants (Fig. 2).

Dorstenia sect. Lomatophora and D. sect. Kosaria form a monophyletic group along with the only species of D. sect. Acauloma sampled (D. barnimiana Schweinf.) and the only African D. sect. Lecania sampled (D. picta). All these taxa are herbaceous or succulent microspermous plants, paleotropical, and usually have seven or more pistillate flowers per receptacle. According to Berg & Hijman (1999)Berg CC & Hijman MEE (1999) The genus Dorstenia (Moraceae). Ilicifolia 2: 1-211., D. sect. Lomatophora and D. sect. Kosaria are close for being caulescent plants with scattered leaves and a mostly supraterraneous stem.

Dorstenia barnimiana, the only species sampled from D. sect. Acauloma (out of three) emerged among the species of D. sect. Kosaria, exactly as Berg & Hijman (1999: 120) inferred from morphological data. These groups are composed of succulent plants and, on the basis of the molecular data presented here, we infer that they should be treated within a single section. This should be better elucidated when the three species of D. sect. Acauloma and other species of D. sect. Kosaria are sampled.

Dorstenia picta, one of the two species of D. sect. Lecania in Africa, emerged from a deep node of the clade D. sect. Lomatophora + D. sect. Kosaria + D. sect. Acauloma and has a very long branch showing high evolutionary divergence. This raises the hypothesis that the African species of D. sect. Lecania can be considered a lineage that is parallel to neotropical D. sect. Lecania. Previous molecular studies also reported the same results (Misiewicz & Zerega 2012Misiewicz T & Zerega NJC (2012) Phylogeny, biogeography and character evolution of Dorstenia (Moraceae). Edinburgh Journal of Botany 69: 413-440.; Zhang et al. 2019Zhang Q, Gardner E, Zerega N & Sauquet H (2019) Long-distance dispersal shaped the diversity of tribe Dorstenieae (Moraceae). BioRxiv 531855. doi: <http://dx.doi.org/10.1101/531855>). Thus, we conclude that these two species (D. picta and D. Subdentata Hijman & C.C.Berg) should be included in another section. Taxonomic studies that focus on African taxa are needed to confirm this.

According to the phylogenetic hypothesis presented in this study, the neotropical species (herbaceous microspermous plants) form a clade nested within the paleotropical species and are sisters, in part, to D. sect. Nothodorstenia (D. elliptica). According to Berg & Hijman (1999)Berg CC & Hijman MEE (1999) The genus Dorstenia (Moraceae). Ilicifolia 2: 1-211., the neotropical species diverged from paleotropical herbaceous plants, event makes by the presence of a bracteate receptacle, being closer to the African D. sect. Nothodorstenia (woody macrospermous plants) than to the other herbaceous sections (paleotropical). Therefore, reproductive traits, such as the presence and absence of bracts in the inflorescence, may play a key role in the evolution of the group, and morphological and anatomical studies focusing on this aspect may help to elucidate the systematics of the group.

Within the neotropical sections (Dorstenia sect. Dorstenia, D. sect. Emygdioa, and D. sect. Lecania), the present analysis indicated that the three sections are polyphyletic and that the neotropical clade can be subdivided into a Central American group and a South American group. Berg (2001)Berg CC (2001) Moreae, Artocarpeae and Dorstenia (Moraceae): with introductions to the family and Ficus and with additions and corrections to Flora Neotropica 7. Flora Neotropica vol. 83. New York Botanical Garden, New York. 346p. stated that it was very difficult to precisely delineate the three neotropical sections. Carauta (1978)Carauta JPP (1978) Dorstenia L. (Moraceae) do Brasil e países limítrofes. Rodriguésia 29: 71-73. used habit, leaf, stipules, and inflorescence shape to subdivide the sections, but molecular biology is questioning whether these characters clearly unify the neotropical monophyletic groups. Thus, in view of the lack of a reliable morphological distinction and the polyphyletic relationships among the species of D. sect. Dorstenia, D. sect. Emygdioa, and D. sect. Lecania, we propose the later two as synonymous of D. sect. Dorstenia.

Taxonomic treatment

Dorstenia sect. Dorstenia L., Sp. Pl. 121. 1753. Type species. Dorstenia contrajerva L. = Sychinium Desv., Mém Soc. Linn. Paris 4: 216. 1826. Dorstenia sect. Sychinia (Desv.) Carauta, Bradea 2(21): 151. 1976. Type species. Sychinium ramosum Desvaux, syn. nov.

= Dorstenia sect. Lecania Carauta, Bradea 2(21): 151. 1976. Type species. Dorstenia turnerifolia Fisch. & C.A. Mey., syn. nov.

= Dorstenia sect. Emygdioa Carauta, Bradea 2(21): 151. 1976. Carauta, Rodriguésia 29(44): 105. 1978. Type species. Dorstenia brasiliensis Lam., syn. nov.

Herbs to subshrubs, monoecious; non cactiform; stems supraterranean to entirely subterranean; internodes elongate or short. Leaves spirally alternate, stipules present, subfoliaceous to subulate. Inflorescences usually bisexual and axillary, mostly bracteate, fringe present; flowers connate; interfloral bracts lacking (occasionally rudimentary); staminate flowers among pistillate ones or at the periphery of the flowering face; pedicellate; tepals 2–3(–4), stamens 2–3, inflexed in the bud, pistillode occasionally present; pistillate flowers sessile, perianth tubular, free or sessile, stigma 2, usually unequal. Fruit dehiscent drupelet, exocarp white and fleshy, turgid, ejecting the endocarp when mature (dry); seed small, endosperm present.

The species are distributed in North America (Mexico) to South America (Argentina), with fewer species in the Amazon basin. Fifty six species are listed in D. sect. Dorstenia. Included species: D. albertorum Carauta, D. appendiculata Miq., D. arifolia Lam., D. aristeguietae Cuatrec., D. asaroides Gardner, D. bahiensis Klotzsch ex Fisch. & C.A.Mey., D. belizensis C.C.Berg, D. bonijesu Carauta & C.Valente, D. bowmaniana Baker, D. brasiliensis, D. brevipetiolata C.C.Berg, D. caimitensis Urb., D. carautae, D. cayapia, D. choconiana S.Watson, D. colombiana Cuatrec., D. conceptionis Cuatrec., D. contensis Carauta & C.C.Berg, D. contrajerva, D. crenulata C.Wright ex Griseb., D. dolichocaula Pilg., D. drakena L., D. elata Gardner, D. erythandra C.Wright ex Griseb., D. excentrica Moric., D. fawcetii Urb., D. fischeri Bureau, D. flagellifera Urb. & Ekman, D. gracilis Carauta, C.Valente & D.S.D.Araujo, D. grazielae Carauta, C.Valente & Sucre, D. hildegardis Carauta, C.Valente & O.M.Barth, D. hirta Desv., D. jamaicensis Britton, D. lindeniana Bureau, D. maris C.Valente & Carauta, D. milaneziana, D. nummularia Urb. & Ekman, D. panamensis C.C.Berg, D. peltata Engl., D. peruviana C.C.Berg, D. petraea C.Wright ex Griseb., D. ramosa (Desv.) Carauta, C.Valente & Sucre, D. rocana Britton, D. roigii Britton, D. romaniucii A.F.P.Machado & M.D.M.Vianna, D. setosa Moric., D. stellaris Al. Santos & Romaniuc, D. strangii Carauta, D. tentaculata Fisch. & C.A.Mey., D. tenuis Bonpl. ex Bureau, D. tuberosa C.Wright ex Griseb., D. turnerifolia, D. umbricola A.C.Sm., D. urceolata Schott, D. uxpanapana C.C.Berg & T.Wendt, D. vitifolia Gardner.

Excluded taxa: Dorstenia picta and D. subdentata.

Dorstenia picta (African species) was excluded from the D. sect. Dorstenia (neotropical taxon) because it does not share the most recent common ancestor with the taxa included in this section. This species emerged from within the clade D. sect. Lomatophora + D. sect. Kosaria + D. sect. Acauloma according to our results. Based on the morphological analysis, we concluded that it is a member of the section D. sect. Lomatophora, mainly because it has an herbaceous habit with creeping to ascending leafy stems and a partly subterraneous stem, which is very similar to D. psilurus Welw. Dorstenia subdentata was not sampled in the phylogenetic analysis, but after undertaking a morphological study of the species, because it is an ebracteate species, we predicted that it should also be treated as a member of the D. sect. Lomatophora.

Acknowledgements

We are grateful to Paulo Ferreira (Universidade Federal do Rio de Janeiro, in memorian) and Luciana Franco (Instituto de Pesquisas Jardim Botânico do Rio de Janeiro), for providing laboratory facilities; and to Claudio Nicoletti, Deborah Hottz (Instituto de Pesquisas Jardim Botânico do Rio de Janeiro), Jim Solomon (Missouri Botanical Garden), Anderson Machado (Universidade Estadual de Feira de Santana), and Welma Souza (Universidade Federal do Amazonas, Brazil), for providing samples of Dorstenieae. We also thank Marcelo Vianna Senior, for helping us format the images. This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES - Finance Code 001). MDMVF was the recipient of grants from CAPES and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). AFC and VFM were the recipients of grants from CNPq and from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ E_02/2017E_02/2017), and LCP received a grant from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, E-26/202.277/2019 and nº E-26/202.278/2019). The authors are grateful to the handling editor and the reviewers of this manuscript.

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Supplementary Material

See supplementary material at <https://doi.org/10.6084/m9.figshare.16569552.v1>

Publication Dates

  • Publication in this collection
    27 Sept 2021
  • Date of issue
    2021

History

  • Received
    28 Mar 2020
  • Accepted
    27 July 2020
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