Evolutionary relationships and taxonomy of Microtea (Microteaceae), a basal lineage in the core Caryophyllales

Abstract The basal position of the small American genus Microtea within the core Caryophyllales was suggested only recently in accordance with molecular phylogeny. However, the specific relationships within the genus were not traced. The results of our phylogenetic analysis based on the matK chloroplast gene suggest the monophyly of Microtea, and Ancistrocarpus and other related genera should be included in it. Microtea is divided into two major sister clades: clade A consisting of M.glochidiata, M.maypurensis and M.tenuifolia, and clade B comprising M.debilis, M.sulcicaulis, M.scabrida, M.celosioides, and M.papillosa. The nrDNA dataset (ITS), although containing only a limited number of accessions, shows the same species number in clade A, and the remaining species studied (M.debilis, M.scabrida and M.celosioides) form clade B. Subgeneric status is assigned to clades A and B corresponding with the names Microteasubgen.Ancistrocarpus subgen. nov. and Microteasubgen.Microtea, respectively. The diagnostic characters at the subgeneric level are as follows: length of pedicels, number of flowers at each node, number of stamens and styles. A multivariate analysis of 13 distinguishing morphological characters supports the results of phylogenetic analysis. All species have similar pericarp and seed ultrasculpture and anatomy, and they share the reticulate pericarp surface (independent of presence or absence of finger-shaped outgrowths on its surface) and rugose or slightly alveolate seed ultrasculpture. On the basis of morphological characters, we accept 10 Microtea species. A checklist includes a new diagnostic key, morphological descriptions and distribution patterns of each species. Galeniacelosioides is the oldest legitimate name available for the plants previously known as Microteapaniculata, for which the combination Microteacelosioides is validated here. The neotypes of Galeniacelosioides and Microteasprengelii were designated from the collections of Prinz Wied at BR. The name M.foliosa is discussed and finally synonymized with M.scabrida. The lectotypes of Ancistrocarpusmaypurensis (≡Microteamaypurensis), Microteadebilisvar.ovata (=M.debilis), M.glochidiata, M.maypurensisvar.angustifolia (=M.tenuifolia), M.glochidiataf.lanceolata (=M.maypurensis), M.longebracteata (=M.celosioides), M.paniculatavar.latifolia (=M.scabrida), M.portoricensis, M.scabrida, M.sulcicaulis, and Potamophilaparviflora (=M.maypurensis) are designated. Microteasulcicaulis is reported for the first time as native to Bolivia, and M.maypurensis is reported from Indonesia (Java), where it is found as an alien plant with an unclear invasion status.


Introduction
The genus Microtea Sw. was described by Swartz (1788) with one species, M. debilis Sw., native to the Lesser Antilles (the Caribbean). The author placed it within the group "Pentandria-Digynia" due to the pentaphyllous perianth, five stamens, and two styles. A pericarp with distinct echinate outgrowths was reported as another indicative character of Microtea (Swartz 1788). Kunth (1817) described a related genus Ancistrocarpus Kunth with the type species A. maypurensis Kunth, and he pointed out that the main differences between Microtea and Ancistrocarpus belong to the reproductive characters such as the different numbers of stamens (five vs eight, respectively) and styles (two vs four or five) and the shape of the pericarp outgrowths (echinate vs apically hooked). Further new generic and species names were mostly based on the same reproductive characters (Roemer and Schultes 1820, Schrank 1821, Link 1821, but none of the genera allied to Microtea have been commonly accepted, and currently Microtea has been considered a single genus that includes all closely related taxa (e.g., Steudel 1841, Moquin-Tandon 1849, Urban 1885, Walter 1909, Rohwer 1993, Marchioretto and de Siqueira 1998. According to the latest studies (Marchioretto andde Siqueira 1998, Hernández-Ledesma et al. 2015), Microtea comprises 10-12 species distributed in Central and South America. They can be distinguished and classified by life history, presence of bracteoles by each flower, and morphology of the pericarp (e.g., Walter 1909, Marchioretto andde Siqueira 1998). However, many important reproductive traits are still poorly studied in this genus, including the fruit and seed anatomy that has been depicted schematically only for M. debilis (Melikian 1993).
Traditionally, Microtea occupied a provisional position within the core Caryophyllales and has been considered as part of the Chenopodiaceae (Kunth 1817, Walter 1906, Takhtajan 2009), Petiveriaceae (Brown and Varadarajan 1985) or Phytolaccaceae (Moquin-Tandon 1849, Walter 1909, Hutchinson 1926, Nowicke 1968, Behnke 1993, Rohwer 1993, Atha 2004, Zhu and Sanderson 2017. Friedrich (1956) suggested that Microtea may be a connecting link between the Phytolaccaceae and Che-nopodiaceae. Also, Behnke (1993) and Behnke and Mabry (1994) reported that the structures of sieve-element plastids and of the pollen grains deviate from those of the other Phytolaccaceae. The recently combined molecular phylogeny based on the petD and matK regions revealed a distant position of Microtea from both Chenopodiaceae and Phytolaccaceae (Schäferhoff et al. 2009). However, only two Microtea species -M. debilis and M. scabrida Urb. -were included in this molecular analysis (Schäferhoff et al. 2009). Currently, the monophyly of Microtea and the relationships between its species have not been confirmed.
The aims of the present paper are (1) to include more species of Microtea in the molecular analysis in order to clarify the relationships between the species of the genus and to confirm the monophyly of Microtea, (2) to provide new data on the carpological characters as the most diverse and taxonomically important traits, and (3) to provide a new taxonomic description of the genus and better determination of the range of each species.

Carpological studies
Several fruits of all species were taken from the herbaria vouchers deposited in herbaria with the permission of the curators. Seed ornamentation was examined using a scanning electron microscope (SEM) JSM-6380 (JEOL Ltd., Japan) at 15 kV after sputter coating with gold-palladium in the laboratory of Electron Microscopy at the Lomonosov Moscow State University. To restore the soft pericarp tissue prior to scanning electron microscopy, the fruits were dehydrated in aqueous ethyl alcohol solutions of increasing concentration, followed by alcohol-acetone solutions and pure acetone. The seeds did not require a complicated treatment prior to SEM due to the presence of the hard seed coat. The cross-sections of the fruits and seeds were prepared using a rotary microtome Microm HM 355S (Thermo Fisher Scientific, USA). Before sectioning, the seeds were soaked in water:alcohol:glycerin (1:1:1) solution, dehydrated in an ethanol dilution series and embedded in Technovit 7100 resin (Heraeus Kulzer, Germany). The cross-sections were observed using a Nikon Eclipse Ci microscope and photographed with a Nikon DS-Vi1 camera (Nikon Corporation, Japan) at the Department of Higher Plants (Moscow State University).

Phylogenetic analysis
The list of vouchers and their accession numbers is provided in Table 1.

DNA extraction and PCR amplification
The nuclear (ITS) and chloroplast (matK) regions of genomic DNA were used for the phylogenetic analysis. Total DNA was isolated from dried leaves using Invisorb® Spin Plant Mini Kit (Stratec Molecular GmbH, Berlin, Germany). ITS-A (Blattner and Kadereit 1999) and ITS4 (White et al. 1990) primers were used for ITS region amplification, and MatK-1RKIM-f and MatK-3FKIM-r were used for matK region (http://botany.si.edu/projects/dnabarcode/matK_PCR_&_Sequencing_Protocols.pdf ). PCRs were performed using 0.75 units of MyTaq Red DNA polymerase (Bioline, London, UK) in 15 μl of original buffer containing MgCl 2 and dNTPs, with 0.3 μM of each primer and 1 μl of unquantified DNA template. Thermocycling was carried out in TProfessional Basic Thermocycler (Biometra, Göttingen, Germany) using the thermal and cycling conditions as described in Shaw et al. (2007): initial denaturation at 80 °C for 5 min; 30 cycles of 95 °C for 1 min, 50 °C for 1 min, a ramp of 0.3 °C/s to 65 °C and incubation at 65 °C for 4 min, with a final extension step of 65 °C for 5 min. A clean-up reaction with exonuclease I and alkaline phosphatase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to remove unincorporated primers and nucleotides before sequencing. The PCR products were sent to Macrogen Europe (Netherlands) for automated sequencing. The primers used for amplification were also used for the sequencing reactions.

Sequence alignment and phylogenetic reconstruction
Data files were assembled, edited and evaluated using Geneious 8.1 software (Biomatters Ltd, Auckland, New Zealand). Regions of ambiguous alignment were excluded from all analyses. After exclusion of these regions we used 754 characters in the nuclear (ITS) and 828 characters in the chloroplast (matK) analysis. The nuclear and chloroplast data were analyzed separately with MEGA7 software (Kumar et al. 2016) which delivered a maximum likelihood (ML) tree based on the Kimura 2-parameter model (Kimura 1980) with support for nodes measured by bootstrap percentages (N70% considered significant). The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016).

Multivariate analysis
Different Microtea species were classified by group average linkage algorithm of cluster analysis constructed on a Gower similarity matrix (Gower 1971) based on thirteen characters including general morphology (life history, pubescence, leaves) and reproductive traits. This approach recognizes the species grouping based on similar characters, but does not provide a true phylogenetic context. The reliability of grouping was assessed at the level p<0.05 using SIMPROF algorithm (Clarke 1993, Clarke andWarwick 2001). Calculations were performed using PRIMER 6.1.6 statistical software (Clarke and Gorley 2006).

Phylogenetic analysis of matK region
The most representative phylogenetic analysis based on cpDNA (matK) dataset shows the monophyly of the genus Microtea, which is divided into two well-supported clades ( basal position within the core Caryophyllales (Brockington et al. 2009, Schäferhoff et al. 2009, Sukhorukov et al. 2015. The tree based on the matK region that includes these three basal lineages suggests the monophyly of Microtea (Fig. 1).

Phylogenetic analysis of ITS region
Based on the nrDNA tree (Fig. 2

Carpological investigations
In all the species studied the fruit is one-seeded (Fig. 3), and the pericarp surface is reticulate (Figs 4-8, A, B, E, F). The finger-shaped (echinate) outgrowths over the  The pericarp consists of several layers; the cells of the innermost layers are usually filled with tannins ( Fig. 9 A, B). The pericarp outgrowths emerge from the mesophyll and consist of several prosenchymatous cell layers.
The seeds are spherical and black, with a rugose (Figs 4-8C, D, G, H) or slightly alveolate (M. sulcicaulis: Fig. 6D) surface, and are basally inserted on the fruit wall. The seed coat of all species consists of a thick (40-50 μm) exotestal layer, with outer cell      walls much thicker than the arch-like protoplast (Figs 3; 9), and 1-2 barely visible layers of tegmen with bar-thickenings of its cell walls. The cells of the exotesta ( Fig. 9) are dark brown due to the presence of unstructured tannin-like substances, but without additional stalactite-shaped deposits (vertical or oblique depositions of tannins originating from the outer cell walls). The annular embryo occupies a peripheral position in the seed, and is located vertically. The perisperm is abundant.
All Microtea species share the reticulate pericarp surface (regardless of the presence or absence of echinate outgrowths) and the rugose or slightly alveolate seed surface. The fruit and seed structure of Microtea, namely the homocellular pericarp consisting of several layers, seed coat with much thicker testa and barely noticeable tegmen with barthickenings of the cell walls, vertical embryo position in one-seeded fruits, and abundant perisperm, is typical for the core Caryophyllales (Sukhorukov et al. 2015(Sukhorukov et al. , 2018.

Diagnostic characters in Microtea and multivariate analysis
All characters discovered in Microtea species are summarized in Table 2.
The results of cluster analysis of the characters suggest the existence of five significantly different groups within the Microtea, these branches being highlighted in black colour ( (1) corresponds with clade A in the phylogenetic analyses (Figs 1, 2), and this group is clearly distant from the remaining species due to character sets 4, 5, 9 and 10. Cluster (2) comprises the species without bracteoles (character 3, state 0), and cluster 3 unites the rest of the genus with the similar sets of characters 3, 4, 5, 8, 9, 10, and 12.

Taxonomy
Artificial key to the Microtea species   inflorescence a spike or thyrsoid; pedicel inconspicuous or up to 3 mm long; flowers actinomorphic, bisexual, subtended by a hyaline bract and two similar bracteoles, sometimes bracteoles absent; perianth of (4)5 glabrous segments or lobes, green, white or yellowish; stamens (4)5 in alternisepalous position, or 6-8 (in both antesepalous and alternisepalous positions), anthers 0.15-0.30 mm, introrse, thecae globose, pollen grains pantoporate; ovary roundish; style not present or very short, stigmas 2-5; fruit nut-like, single-seeded, dry; pericarp projections (if present) not evident in flowering condition, pericarp at fruiting stage reticulate, mostly having finger-shaped outgrowths (emergences) that can be plumose (with additional smaller hair-like projections) or hooked at their apices; seeds spherical, black, with rugose or alveolate surface, with annular embryo located vertically and abundant perisperm. Ten species distributed in the (sub)tropics of the Americas; two -M. debilis and M. maypurensis -are considered as aliens in the humid tropics of Africa (Cameroon) and Asia (Indonesia), respectively.  (Swartz 1788) that in such cases he used the herbarium collections in Banks's possession (Stearn 1961, Howard andHoward 1982). The collections used by Swartz (1788) are listed in Swartz's subsequent publication, Flora Indiae Occidentalis, in which the original locality of M. debilis was stated more accurately as the island of "St. Eustathii", and "Masson" was indicated as the collector (Swartz 1797: 543). This specimen is the only element associated with the taxon by the original author, and consequently it was designated as the lectotype of the name (Howard and Howard 1982: 76). Although this was not indicated in the protologue, probably a further collection was available to Swartz by that time: a specimen of M. debilis collected by H. de Ponthieu (reportedly in Grenada) was acquired by Swartz from Banks and subsequently given to L.J. Montin, a Swedish collector of herbarium material, whose private herbarium became part of S along with that of Swartz himself (Lindman 1916). Description. Annuals, glabrous; stems decumbent, up to 30 cm (Fig. 11A); rosulate leaves up to 9 (10-12) cm, long-petiolate, obovate or oblong, mostly persistent; cauline leaves rhombic or ovate, cuneate; inflorescence a spike (Fig. 11B); flowers sessile or very shortly pedicellate (pedicels at fruiting ~1 mm); bracteoles absent; perianth segments 5, greenish, lanceolate or oblong; stamens (4)5; stigmas 2, thick; fruit roundish, 1.1-1.25 mm long and 1.0-1.2 mm wide (Fig. 4A, B), with finger-shaped outgrowths (up to 0.4 mm long); seed ~1.0 mm, with rough surface (Fig. 4D).
Habitat. Sands, forest margins, or as a weed; altitudes up to 1000(1200) m a.s.l. Distribution. Native to American tropics (Fig. 12).   St. Thomas, Charlotte Amalie, 9 Feb 1913, N.L. Britton et al. 470 (NYBG01509850 -image!); Note: Data were not available on the presence of the species in Cuba, in agreement with the recent treatment of Microtea in this country (Greuter 2002). As alien found in tropical Africa (Bamps 1974 Walter (1909) as referable to M. paniculata (specimen destroyed at B). Along with the characters stated in the protologue of G. celosioides, this fact provides indirect evidence that Prince Maximilian's collections were the likely source of the introduction of G. celosioides to the Botanical Garden in Berlin. There are no specimens of the original material of G. celosioides in existence, which probably was acquired by B and then destroyed (Stafleu and Cowan 1985). Since the characters of the Prince Maximilian's specimen at BR are in good agreement with the protologue of G. celosioides, we designate this specimen as a neotype of the species name. This name is therefore the earliest one available for the species also known as M. paniculata. =M. paniculata Moq. in DC.,Prodr. 13 (2) paniculata is the length of the bracts that is equal to those of the flowers (Walter 1909). However, the length of the bracts in both taxa is equal to that of the flower buds, and the length of fully opened flowers is greater than the length of the subtending bract. Description. Annuals or biennials; stems erect, up to 100 cm; leaves petiolate (petioles up to 2.0 cm), blades 3.0-10.0(12.0) cm long, 0.2-2.0 cm wide, cuneate, lanceolate to oblong, rarely ovate, glabrous or their margins and mid-rib below covered with papillae; inflorescence a spike, long and spreading, whip-like; flowers with a bract and two filiform bracteoles (often not well-visible), bracts longer than flowers at the beginning, then equal to the perianth segments; pedicels up to 1.0 mm at fruiting, perianth segments 5, oblong or ovoid; stamens 5-8; stigmas 2, thick; fruit with scattered short outgrowths, fruit body (1.0)1.1-1.4 × 0.9-1.1 mm, 1.5-2 times as long as the perianth (Fig. 4E, F); pericarp readily scraped off the seed; seed ~1.0 mm, with rough surface (Fig. 4H).

M. celosioides
Habitat. Forest margins, roadsides, river banks, on sandy and rocky substrates; altitudes up to 1000 m a.s.l.
Habitat. Rocky and sandy substrates at altitudes up to 1500 m a.s.l. Distribution. Subtropical South America (Fig. 19).    fig. 63 in Schrank (1821). (Fig. 22). Note: von Ledebour (1821) intended (Art. 6.11) to introduce his new species name as a replacement name for the later homonym published by Schrank, and both names are therefore necessarily homotypic. Schrank (1821) published his new species names based on seeds sent or brought from Brazil by C.F. von Martius. Original herbarium collections of Potamophila parviflora Schrank were not found at M and most likely had never been prepared (H.-J. Esser, pers. comm. 2018), and the illustration published as part of the protologue is the only original element available for lectotypification (Art. 9.4). The protologue of Potamophila parviflora indicates that the new species has five styles (although only three are visible in the accompanying illustration) and a pericarp with setae. Coupled with pedicellate flowers and petiolate leaves, easily recognizable in the illustration, these characters are indicative of Microtea may-purensis, of which P. parviflora (Ancistrocarpus schrankii) is a later synonym. This agrees with the conclusions of Moquin-Tandon (1849), Walter (1909) and Nowicke (1968). Schrank (1821) stated that the new species was collected in "Brasilia prope sinum Omnium Sanctorum ad flumina locis umbrosis". There is a gathering, Herb. Martius 2198 (M 0274659, M 0274661, M 0274662), which was collected by Martius in Bahia, Cachoeira, along the Paraguaçu River at the distance of ca. 35 km from Baía de Todos os Santos (Bay of All Saints), in December 1818 (see the route of Martius' expedition in Tiefenbacher (1983)). This is the likely type locality of P. parviflora, and the gathering may be the voucher for the seed collection sent by Martius to the Munich Botanical Garden. Description. Annual or biennial, glabrous; stems erect, up to 60 cm, branched; rosulate leaves oblong, usually withered, lower leaves oblong or spatulate, cuneate, petiolate (petioles up to 2.5 cm), 3.0-8.0 cm long and (0.2-0.4)0.5-2.0 cm wide (sometimes narrower), acuminate; inflorescences a spike, not one-sided, often spreading; flowers solitary (rarely two per node), with a bract and two bracteoles, pedicellate (pedicels 1.5-3.0 mm), perianth segments 5, oblong to ovoid, white or yellowish; stamens 5-8, stigmas 3-5, filiform; fruit slightly protruding from the perianth or up to twice its length, fruit body 1.0-1.1 mm across, with outgrowths 0.2-0.5 mm long terminating in a group of 2-4 hooked hairs (Fig. 8A, B); pericarp readily scraped off the seed; seed ~1mm, with rough surface (Fig. 8D).