A New Species of Mimosa L. ser. Bipinnatae DC. (Leguminosae) from the Cerrado: Taxonomic and Phylogenetic Insights

Mimosa carolina (Leguminosae), a new species from the Parque Nacional Chapada das Mesas, located in the northern limit of the Brazilian Cerrado, is described and illustrated. In addition, a phylogenetic reconstruction was performed to recover the position of this species in relation to the main clades of the genus Mimosa. This new species is assigned to sect. Habbasia ser. Bipinnatae and exhibits relevant morphological differences with all described species of this series, most notably the prostate habit, glabrous stems, and absence of internal spicules. Our results indicate that this new entity is clearly nested in a strongly supported clade with other striated-corolla species of ser. Bipinnatae. Therefore, ser. Bipinnatae appears to be monophyletic, and a morphologically and ecologically cohesive group within Mimosa. An updated identification key for this series is provided.


Introduction
The large genus Mimosa (Leguminosae) is particularly diverse in the Cerrado, a savanna-dominated ecoregion covering 2 million square kilometers in central Brazil and eastern parts of Bolivia and Paraguay [1,2]. Vegetation in the Cerrado varies from tall savanna woodlands to open grasslands, with gallery forests or seasonally waterlogged marshes and fields occurring along watercourses. Soils are generally nutrient-poor, highly acidic and aluminum-rich, and precipitation is highly seasonal with a pronounced winter dry season from May to October [2]. High seasonality and the accumulation of dry biomass makes Cerrado savannas and grasslands fire-prone, and species from many plant lineages show adaptations to cope with fire regimes [3].
The detailed monograph of Barneby [4] for the Neotropics and latter studies [5][6][7][8][9][10][11][12] allowed the recognition of ca. 200 species of Mimosa in the Cerrado. Most of these species exhibit a restricted distribution; consequently, this ecoregion exhibits high levels of endemism for the genus, particularly in the highlands [13]. sect. Mimosa. However, new phylogenetic insights are challenging this proposal; in these works, based on different chloroplast markers [14,15], only sect. Mimadenia was recovered as monophyletic. Members of sections Batocaulon and Habbasia formed a paraphyletic clade, whereas sect. Calothamnos was nested within sect. Mimosa.
In spite of the discussion about the sections proposed by Barneby [4] several infrageneric groups of the genus at the level of series or subseries were recovered as monophyletic, such as sect. Batocaulon ser. Stipellares Benth., sect. Mimosa subser. Obstrigosae (Benth.) Barneby, and sect. Habbasia ser. Bipinnatae [14,15]. However, for many infrageneric groups, taxonomic sampling needs to be increased to allow for more robust conclusions.
Detailed studies to infer diagnostic characters for well-supported groups in Mimosa are still lacking. A preliminary assessment of character evolution in Mimosa has shown that, except for extrafloral nectaries and pollen grain morphology, the main characters used to define infrageneric groups are homoplasic [15]. The fact that many morphological characters evolved independently in different clades makes the search for diagnostic characters a challenging task in Mimosa.
The aim of this work was to evaluate a possible new Mimosa species from the Cerrado, as well as investigate its phylogenetic position in the genus [15]. In addition, we re-evaluated the monophyly of ser. Bipinnatae with the inclusion of newly generated sequences in the phylogeny.    Mimosa carolina is similar to the widespread M. somnians Humb. & Bompl. ex Willd, from which it differs by a prostrate habit with slender stems (vs. erect subshrub with robust stems), that are glabrous (vs. pubescent to hispid, rarely glabrous), and unarmed (vs. sometimes armed), shorter pinnae (4-8 mm long) with 6-10 pairs of leaflets (vs. pinnae 10-55 mm long with 13-50 pairs of leaflets), the insertion of pinnae on leaf rachis v-shaped (vs. insertion of pinnae straight on the leaf rachis), and the absence of interpinnal spicules (vs. interpinnal spicules present); it differs from the poorly known M. leptorhachis Benth. by the prostrate habit (vs. erect), glabrous stems (vs. hispidous), insertion of pinnae on leaf rachis v-shaped (vs. insertion of pinnae straight on the leaf rachis) and absence of interpinnal spicules (interpinnal spicules present). In addition, it differs from M. brachycarpa Benth. by its prostrate habit (vs. erect 1-2 m tall), petioles which are 25-45 mm long (vs. 1-10 mm long) and larger (36-40 mm long), and glabrous fruits (vs. fruits 12-18 mm long, densely setose).

Distribution and Ecology
At present, this species is only known from the Parque Nacional Chapada das Mesas, in Maranhão (Brazil), in the northern extreme of the Cerrado ( Figure 3). This region has not been adequately prospected by botanists [16]; it explains why this entity has remained unknown. It was found growing on deep, sandy soils in sparse savanna vegetation at 280 m elevation, about sea level.

Distribution and Ecology
At present, this species is only known from the Parque Nacional Chapada das Mesas, in Maranhão (Brazil), in the northern extreme of the Cerrado (Figure 3). This region has not been adequately prospected by botanists [16]; it explains why this entity has remained unknown. It was found growing on deep, sandy soils in sparse savanna vegetation at 280 m elevation, about sea level.

Etymology
The epithet refers to Carolina, the municipality of Maranhão State in Brazil where the collection site is located.

Conservation notes
Mimosa carolina is only known from the type locality at the Parque Nacional Chapada das Mesas (160,000 hectares). It was recorded as locally abundant in a collecting site in the southern portion of the national park (M. Simon pers. observation). According to the revision of specimens in herbaria

Etymology
The epithet refers to Carolina, the municipality of Maranhão State in Brazil where the collection site is located.

. Conservation notes
Mimosa carolina is only known from the type locality at the Parque Nacional Chapada das Mesas (160,000 hectares). It was recorded as locally abundant in a collecting site in the southern portion of the national park (M. Simon pers. observation). According to the revision of specimens in herbaria (Supplementary material) in recent field trips, this species was not found in other localities. Considering that the region is poorly collected, we prefer to classify M. carolina as Data-Deficient (DD) according to the IUCN criteria [17].

Morphological and Phylogenetic Analyses
The taxonomic identification and morphological characterization of a set of more than 100 specimens from ser. Bipinnatae (Supplementary material) allowed the recognition of a new entity among recent collections from Maranhão State in Brazil. This new entity, M. carolina, was assigned to ser. Bipinnatae based on the presence of diplostemonous, tetramerous flowers with a plurinerved and striate corolla. This new species exhibits differences in several characters compared with other members of the ser. Bipinnatae (Table 1). It can be recognized by a combination of characters, some of them shared with other species from ser. Bipinnatae: prostrate habit with slender stems growing from a woody xylopodium; stems glabrous, unarmed; pinnae rachis 4-8 mm long bearing 6-10 pairs of leaflets; insertion of pinnae on leaf rachis v-shaped; and absence of interpinnal spicules (Figures 1 and 2).  Barneby [4]).
The present phylogenetic analysis, based on the plastid trnD-trnT region, included nine species, two subspecies and four varieties of ser. Bipinnatae, as well as three species from other series of sect. ser. Bipinnatae based on the presence of diplostemonous, tetramerous flowers with a plurinerved and striate corolla. This new species exhibits differences in several characters compared with other members of the ser. Bipinnatae (Table 1). It can be recognized by a combination of characters, some of them shared with other species from ser. Bipinnatae: prostrate habit with slender stems growing from a woody xylopodium; stems glabrous, unarmed; pinnae rachis 4-8 mm long bearing 6-10 pairs of leaflets; insertion of pinnae on leaf rachis v-shaped; and absence of interpinnal spicules (Figures 1,2).
Other differences with species of Bipinnatae are: 1) from M. brachycarpa, by the lax foliage and leaves with longer petioles (vs. crowded foliage along stems and mainly subsessile leaves) and a higher number of ovules; 2) from M. monacensis Barneby (Figure 1). We performed a key for identification of all species of the series Bipinnatae (Table 1).
The present phylogenetic analysis, based on the plastid trnD-trnT region, included nine species, two subspecies and four varieties of ser. Bipinnatae, as well as three species from other series of sect.

Discussion
Based on morphological evidence, Mimosa carolina could be readily assigned to ser. Bipinnatae because of the presence of a striate corolla, a distinctive characteristic that defines this group [4], with few exceptions. M. carolina can be recognized by a combination of characters, some of them shared with other species from this series: a prostrate habit with slender stems radiating from a xylopodium; stems glabrous, unarmed; pinnae 4-8 mm long with 6-10 pairs of leaflets, insertion of pinnae on leaf rachis articulated forming a v-shape, and the absence of interpinnal spicules. The habit and absence of interpinnal spicules are two character states that seem to be exclusive to M. carolina, since most species of ser. Bipinnatae are erect subshrubs without prostrate stems, and interpinnal spicules are present in all species of the series [4]. The singular shape of the pinnae rachis of M. carolina, forming a V-shape after the pulvinule, is not found in the majority of taxa of ser. Bipinnatae, excepting M. somnians var. leptocaulis (Benth.) Barneby; the latter has a pinna rachis that forms a little angle after its insertion [4].
Although M. leptorhachis (erroneously annotated as "Mimosa leptorachis" by Barneby [4] (p. 463)) resembles M. carolina, the observation of the type of the former allowed us to consider them different species, due to the erect habit with robust stems and presence of interpinnal spicules in M. leptorhachis. In fact, the identity of M. leptorhachis is doubtful since it is only known from the type collection, which lacks carpological information [4,18], and no more specimens appear to be available in herbaria to allow a proper assessment of its rank and status.
Comparisons of sequence variation based on the trnD-trnT locus shows that M. carolina is genetically distinct from closely related taxa, reinforcing its classification as a separate species. The results of our phylogenetic reconstruction show that the new species is nested with all other representatives of ser. Bipinnatae, which formed a highly supported clade (PP = 1), in line with the morphological classification of Barneby [4]. Our increased sampling within this group (nine additional taxa) confirms previous results based on a smaller sampling (three species; [15]) and reinforces ser. Bipinnatae as a phylogenetically and morphologically cohesive group. This consistent infrageneric group now contains 13 species and 15 varieties, including taxa listed by Barneby [4], as well as M. carolina, described here. This series is distributed mainly in the Brazilian Planaltine and Guayana Highlands; only M. somnians has a wider distribution area, ranging from southern Mexico to northeastern Argentina [4]. Here, we did not include M. trinerva V.F.Dutra and F.C.P.Garcia, which was provisionally assigned to ser. Bipinnatae [6], since analysis of herbarium specimens indicates that it would be more properly placed in ser. Pachycarpae Barneby.
Our new phylogeny also included samples of four infraspecific taxa of M. somnians, which did not form a monophyletic clade. The divergence between infraspecific taxa of M. somnians, which appeared in different, well-supported clades, suggests that they might be better interpreted as distinct species. Indeed, this species is morphologically highly diverse and configures a taxonomic complex with four subspecies and ten varieties [4]; some of them were originally described as species [17] but later treated as varieties by Barneby [4].
The differentiation between infraspecific taxa of M. somnians in the literature has been based, in some cases, on quantitative traits with substantial overlap, making it difficult to separate them (e.g., M. somnians subsp. longipes (Barneby) Barneby; [4]). In other cases, diagnostic characters are discrete and allow a sharp differentiation. This is the case of M. somnians var. lupulina (Benth.) Barneby, which is easily separated from other varieties of M. somnians by its dilated bracts [4]. Incidentally, the close relationship between M. somnians var. lupulina and M. brachycarpa, which were recovered in our phylogeny as sister species, is supported by the presence of a set of wide external floral bracts in the inflorescence, which is shared by both taxa.
Overall, our phylogenetic analysis contributed to the understanding of the relationships between ser. Bipinnatae taxa, showing that Mimosa species with striate, plurinerved corollas comprise a genetically cohesive group, and infraspecific taxa under M. somnians do not form a monophyletic clade and their taxonomic status deserves future investigation. However, a more representative taxon sampling will be needed to better access relationships within ser. Bipinnatae, since only nine out of the 27 currently recognized taxa [4,7] have been sampled to date. Likewise, investigating species boundaries in more detail would require multiple accessions for each species and sequencing of highly variable loci that allow discrimination between species.
It is interesting to point out that the Chapada das Mesas National Park is one of the largest units of conservation in the Cerrado. Recent expeditions and taxonomic work resulted in the description of other endemic species from this location (e.g., Philcoxia maranhensis Scatigna, [19]; Dyckia maranhensis Guarçoni & Saraiva, [20]). Therefore, discovering new endemic species here reinforces the conservation value of this unit in the Biodiversity Hotspot of the Cerrado.

Materials and Methods
More than 100 specimens of ser. Bipinnatae from the following herbaria: BAB, CEN, CTES, LIL, MBM, MO, NY, RB, SI, SP, UB and UFG, were revised. This set of specimens comprised all taxa of the series covering their complete area of distribution (Supplementary material). The taxonomic identification was checked according to Barneby [4] and their morphological characters were measured and/or registered. This revision included the nomenclatural types of taxa and synonyms, as well as the images available from different herbarium databases: JSTOR [21], Kew Botanic Gardens [22], The Barneby Legume Catalogue [23], TROPICOS [24], and SpeciesLink [25].
Several field trips across the Cerrado in the Brazilian states of Goiás, Maranhão and Tocantins were carried out between 2016 and 2019 to collect specimens of ser. Bipinnatae. Samples of leaves for DNA extraction were also collected in these expeditions, which were dried in silica gel and stored at −18 • C in the EMBRAPA laboratory in Brasilia.
DNA was extracted using the protocol of Inglis et al. [26], which includes a pre-wash treatment with sorbitol to remove interfering metabolites. DNA was quantified using Nanodrop Nuclei Acid Quantification ® (Thermo Fisher, Waltham, MA, USA) and agarose gel to check integrity and concentration.
We based our phylogenetic analysis on the plastid trnD-trnT region, which was previously used to infer the phylogeny of Mimosa [10,15]. Amplification of the trnD-trnT region followed the same PCR protocol and primers (trnD2, trnE, trnT, and trnY), as described in Simon et al. [14]. Sequencing reactions using successfully amplified products were performed with the four primers using the Big Dye Terminator kit ver. 3.1 (Applied Biosystems, Foster City, CA, USA).
Consensus sequences from the four sequence strands were assembled using Geneious (v. 6.0.6, Biomatters Ltd.). Sequences generated in this work and those obtained from GenBank were aligned using Clustal W v. 2.1. [27] under default parameters. The aligned trnD-trnT dataset was composed of 12 terminals and 1495 bp. Bayesian analysis was carried out with MrBayes, version 3.2.2 [28], using the GTR + I + G nucleotide substitution model, which was the best model selected in jModelTest v. 2 [29].
We performed two runs in parallel of four Markov chain Monte Carlo for 10 6 generations, with trees sampled every 5000 generations. Permutation of parameters was initiated with a random tree and four simultaneous chains set at default temperatures. Convergence of runs was assessed by inspecting whether the standard deviation of split frequencies of runs was less than 0.01, and the first 25% of the trees were discarded as burn-in. Trees sampled from post-burn-in were summarized into a 50% majority-rule consensus tree that included posterior probabilities (PP).
Voucher information, taxon authority, and GenBank accession numbers of newly generated sequences, as well as those of sequences published in other studies used in our analysis, are provided in Table 2. The aligned trnD-trnT dataset and trees generated in Bayesian analysis are available in the TreeBASE repository [30]. Table 2. Voucher information, locality, and GenBank accession numbers of taxa used in the phylogenetic analysis.

Taxon
Voucher (Herbarium) Locality GenBank Accession Number Source