The monotypic Brazilian genus Diacrodon is a synonym of Borreria ( Spermacoceae , Rubiaceae ) : morphological and molecular evidences

Diacrodon is a monotypic genus of the tribe Spermacoceae (Rubiaceae), endemic to northeastern Brazil. Diacrodon compressus is frequently misidentified with a two lobed calyx species of Borreria, B. verticillata. Traditionally, in Spermacoceae the fruit type was considered a diagnostic character among the genera. In this sense, D. compressus presents a strongly compressed, one seeded and indehiscent fruit (vs. globose, two seeded and dehiscent fruit in B. verticillata). In this work, we address two objectives: evaluate the systematic position and determine the identity of Diacrodon in respect to other taxa. Molecular analyses using ITS and ETS indicate that D. compressus is strongly related to other species of Borreria. The morphological results revealed that D. compressus, despite of its type of fruit, is identical to Borreria in floral and palynological features. As conclusion, the new combination Borreria diacrodonta is made and a lectotype is designated. An updated description of the species and a key to the Borreria species with a two lobed calyx are provided. The distribution of B. diacrodonta is extended to Brazilian states Goiás and Minas Gerais, and Paraguay. By this taxonomical change it has become clear that the dehiscence of the fruits lack taxonomic value in the delimitation of Borreria.


INTRODUCTION
Diacrodon is a monotypic genus that is endemic to Brazil.Its sole species Diacrodon compressus Sprague was described from two dried specimens that were collected by Bolland in Ceará, Brazil (Sprague 1928).Since its description, Diacrodon was recorded in species lists only for the original locality (Robbrecht 1988, Andersson 1992).Recently, Delprete and Jardim (2012) and Souza (2015) included D. compressus in new Brazilian floristic works.Moreover, Diacrodon is scarcely treated in other bibliographic manuscripts probably due to its very restricted distribution and its large similarity with Borreria verticillata (L.) G. Mey., with which it is often confused.
In the original description, Sprague mentioned that the specimens analyzed resemble B. verticillata based on general aspects as well as for the occurrence of flowers with a two lobed calyx.Morphologically, Diacrodon has been defined primarily based on its fruits, described as strongly compressed, indehiscent and single-seeded.In this sense, fruit dehiscence was classically considered a character with taxonomical value to separate genera in the Spermacoceae tribe.Nowadays however, it is a questioned treat in the delimitation of related genera such as Borreria and Spermacoce.Dessein (2003) treated Diacrodon as part of the Borreria-Spermacoce complex and argued that the difference in morphology of the fruits is a result of a recent evolutionary event, also he mentioned that this character is not enough to treat Diacrodon as a separate genus.
In South America, several taxonomic and morphological studies recognize Spermacoce and Borreria as two separate genera mainly by floral and palynological characters which are valuable to distinguish Borreria from Spermacoce (Miguel andCabral 2013, Florentin et al. 2016).They also mention that fruit dehiscence is not a valuable character to delineate the two genera.Moreover, they indicate that Borreria can be distinguished by a set of characters such as axillary glomerules (with bilateral development, completely rounding the stem), flowers with exserted stamens and stigma, campanulate, infundibuliform, ciatiform or subhypocrateriform corolla, and spheroidal or oblate-spheroidal, porate, colporate or colpate with endocingulum pollen grains, and tectate perforate exine (Miguel and Cabral 2013, Miguel 2016, Sobrado 2016).
As part of a larger study on Borreria and because Diacrodon is often compared with one of its species, we address two objectives in this work: (1) evaluate the systematic position of Diacrodon with respect to other genera of the tribe Spermacoceae, and (2) determine the identity of D. compressus.For this reason, we analyze the phylogenetic position of Diacrodon using ITS and ETS sequence data.Additionally, we study flower, fruit, seed and pollen morphology of Diacrodon and make a comparison with two morphologically similar species of Borreria that also have a two lobed calyx (B.spinosa Cham. & Schltdl. ex DC., and B. verticillata).

TAXONOMICAL AND MORPHOLOGICAL STUDIES
Herbarium material was consulted at ASE, CTES, HUEFS, K, NY, RB, SI, UB, UEC, UFRN and US (Herbarium acronyms follow Thiers 2016).Measurements, colors and other details given in the descriptions are based on herbarium specimens.We used conventional taxonomic methods.
In order to analyze the fruit morphology of D. compressus using LM, fruits were fixed in FAA (formaldehyde, acetic acid, 70% alcohol, 90:5:5) and subsequently dehydrated using tertiary butyl alcohol series.After dehydration, fruits were embedded in paraffin (Johansen 1940), transversely sectioned with a rotary microtome, and finally stained with safranin-astra blue (Luque et al. 1996).

MOLECULAR STUDY
In total 55 ingroup taxa are included in the molecular analysis.Bouvardia ternifolia (Cav.)Schltdl. is used as outgroup taxon.The ingroup contains representatives of 14 genera of the Spermacoce clade (Kårehed et al. 2008, Salas et al. 2015) of which the majority has been analyzed before (Kårehed et al. 2008, Groeninckx et al. 2009, Neupane et al. 2015, Salas et al. 2015).For this study, we included representatives of Diacrodon compressus and increased the sampling of the American Borreria species from 8 to 12. Selected DNA markers for this study are the nuclear ribosomal ITS and ETS.Methods used for DNA extraction, PCR amplification, sequencing and alignment follow Janssens et al. (2006Janssens et al. ( , 2007Janssens et al. ( , 2016)).Model selection for the Bayesian inference analysis was conducted with ModelTest 3.06 (Posada and Crandall 1998) under the Akaike Information Criterium (AIC).The GTR+G model was selected for both ITS and ETS.Maximum Likelihood analyses were conducted using the RAxML search algorithm (Stamatakis et al. 2005) under the GTRGAMMA model (Stamatakis 2006).Support values for the best-scoring ML tree were obtained by analyzing five hundred bootstrap trees using the RAxML Rapid bootstrap algorithm (ML-BS).Appendix provides a list of all taxa analyzed with inclusion of localities, voucher information and GenBank accession numbers.
Diacrodon compressus falls into a clade together with American Borreria species and is strongly supported (Clade I, ML-BS=87).In this large clade, we can distinguish two subclades.The first subclade subclades (IA and IB) is B. verticillata, another species with a two lobed calyx but which is not closely related to D. compressus.
Additionally, the species of the related genus Spermacoce are divided in two clades.Clade III, containing species of the related genus Spermacoce, is strongly supported (ML-BS=95) and consists of two subclades.One of this subclades contains the American Spermacoce taxa, S. confusa Rendle, S. glabra Michx., S. tenuior L., and the only Australian species included in this work, S. breviflora F. Muell.ex Benth.Whereas the other clade consists of the American species, S. eryngioides (Cham.& Schltdl.)Kuntze, S. incognita (E.L. Cabral) Delprete, and S. prostrata Aubl.The latter species were initially treated under Borreria sect.Pseudodiodia (sensu Bacigalupo and Cabral 1996), but recently they were designated as Spermacoce (Florentin et al. 2016).Furthermore, this clade is related to species of Psyllocarpus yet only with moderate support (ML-BS=67).

MORPHOLOGICAL ANALYSES
Morphological characters were compared between Diacrodon compressus and either the species it was formerly confused with, Borreria verticillata, as well as the species it is closely related to, B. spinosa (Table I).
Flower morphology (Fig. 2) Except for the campanulate corolla and the spatulate calyx lobes in B. verticillata (vs.infundibuliform and narrowly triangular respectively in D. compressus) there are no significant differences in the morphological structures of the flowers between these similar species (Fig. 2a-d, i-l).On the other hand, B. spinosa differs mainly in the calyx lobes indumenta, the morphology and papillae coverage of the stigma, and the form and length of the nectariferous disc cells (Fig. 2e-h).
Fruit morphology (Fig. 4a-h) Fruit morphology (fruit form and dehiscence pattern of capsules and valves) can be used to distinguish the three species investigated here.Diacrodon compressus can be easily distinguished from Borreria spinosa and B. verticillata by having cuneiform to obovate capsules and strongly compressed to the septum (Fig. 4a).B. spinosa and B. verticillata present ovate to narrowly obovate and globose or subglobose capsules (Fig. 4g, h).Also, D. compressus has indehiscent fruits, however sometimes the capsules split tardily into two indehiscent valves (mericarps).Transverse sections reveal a region between the two locules where the septum cells are thinner, indicating the area where the dehiscence occurs (Fig. 4f).In comparison, the capsules of B. spinosa and B. verticillata show always the same dehiscence pattern, except when the valves split.In B. verticillata both valves can be dehiscent (Fig. 1h), resembling B. spinosa (Fig. 4g), yet can also present two indehiscent valves similar to D. compressus (Fig. 4a); but in the last case the capsule and seed are not compressed.Moreover, the seeds always undergo a normal development in each locule in both Borreria species analyzed here.To date, it was always assumed that D. compressus as one seeded (Sprague 1928).However, SEM and LM images of transverse sections of fruits reveal the presence of capsules with one normal and one aborted seed as well as capsules with two normal seeds (one per locule) [Fig.4b-f].
Seed morphology (Fig. 4i-k) In spite of the similarities in seed morphology between the three species, Diacrodon is somehow distinct as it is characterized by medium sized seeds, which are laterally compressed and narrowly elliptic in outline (ventral view).Seeds of B. spinosa and B. verticillata are elliptic to obovate, whereas B. spinosa has larger seeds and B. verticillata has smaller seeds.In addition, Borreria species have seeds with a wider longitudinal groove covered by strophiole compared to those of Diacrodon.

PHYLOGENETIC POSITION OF Diacrodon
Our phylogenetic analysis supports the inclusion of Diacrodon in Borreria in order to form a monophyletic and natural group.Its sole species, D. compressus, is intermingled in a group with some American Borreria species.In agreement with previous results, we found that Borreria currently represent a polyphyletic genus.Dessein (2003) and Salas et al. (2015) obtained similar results, based on a more limited number of American Borreria species.First, some species that currently are Borreria subsect.Latifoliae (with deeply bifid stigma) fell in their study in a different and well supported clade indicating that this group is probably a monophyletic clade (Sobrado 2016).Second, in their results others American Borreria species  (with capitate, bilobed or slightly bifid stigma) which are placed in Borreria subsect.Borreria formed a clade with African species of the related genus Spermacoce.However, this last relation is refuted by our study, because African Spermacoce representatives form a strongly support clade, which is weakly related to Ernodea and Hexasepalum.In addition, American and Australian Spermacoce species are nested in a different monophyletic group related to Psyllocarpus and distinct to Borreria.
Although, we consider that these results partially elucidate the controversy regarding the generic status between Borreria and Spermacoce, we believe that it is important to find synapomorphies that can support a generic rearrangement.To further assess the generic limitation of Borreria and Spermacoce, and the relation between American, Australian and African species, a more thorough morphological and molecular study that includes more gene markers and a larger sampling is needed.

IDENTITY OF Diacrodon compressus
Most of the morphological characters analyzed in this study support the inclusion of Diacrodon compressus in the genus Borreria.
Distinction of Diacrodon into a genus of its own was based on the lack of dehiscence in the fruit and on the development of a single seed per fruit.In this sense, the presence of completely indehiscent capsules corresponds with the fruit morphological description of some Spermacoce species.Also, we notice that Sprague´s species has a tardily dehiscent capsule opening in two indehiscent valves.However, this type of dehiscence should be evaluated whether it is an adaptation to flooded environments because some specimens of Diacrodon were recorded to grow on margins of streams and rivers.This hypothesis could be supported by the fact that some species of Borreria which also have capsules with both valves being indehiscent are occurring in similar conditions [eg. B. dasycephala, B. schumannii and B. hyssopifolia (Willd. ex (Cabral et al. 2011).Even Dessein (2003) observed a tendency to abort one of the ovules in Spermacoce hockii (De Wild.)Dessein.One Australian species, S. gibba Harwood is also characterized by an indehiscent and one-seeded capsule (Harwood and Dessein 2005).These examples show that the fruit characteristics referred by Sprague are not exclusive to Diacrodon.Also, it is according with that fruit dehiscence is not valuable character to differentiate Borreria from Spermacoce.
However, Borreria is currently defined by a distinct set of floral and palynological characters (Miguel 2016, Miguel andCabral 2013).In this sense, Diacrodon compressus with its flowers with exserted stamens and stigma, infundibuliform corolla, oblate-spheroidal, colporate pollen grains, and tectate perforate exine, is identical to Borreria.In addition, the shape of the stigma that defines Borreria subsect.Borreria, is capitate, bilobed or shortly bifid.According to Diacrodon is characterized by a short bifid stigma as in subsection Borreria, which also includes others species with calyx two lobed.Pollen morphology in Diacrodon, B. spinosa and B. verticillata corresponds to the pollen type III described by Pire (1996), which is exclusively found among American Borreria species.
In conclusion, by combining morphological and molecular evidence we suggest to transfer the monotypic genus Diacrodon to the species-rich genus Borreria.This taxonomical change implies a different fruit morphology for Borreria, and makes clear that the dehiscence of fruits alone contains no taxonomic value to delineate Borreria from Spermacoce.Both genera have at least 4 types of fruits: (1) Capsules  5 and 6a, b.

Figure 1 -
Figure 1 -ML tree showing phylogenetic position of Diacrodon in Spermacoce clade based on nuclear (ITS, ETS) data.Values at the nodes represent bootstrap support (ML-BS).

Figure 2 -
Figure 2 -Flower morphology.a-d.Diacrodon compressus, a. Flower bud.Note the narrowly triangular calyx lobes and the hypanthium pilose in the upper part; b.Shortly bifid stigma, showing short papillae at the inner surface; c.Nectariferous disc; d.Isodiametric and uniform disc cells; e-h.Borreria spinosa, e. Flower bud.Note the narrowly triangular calyx lobes and the hypanthium puberulous in the upper part; f.Bilobed stigma, showing fully surface covered with papillae; g.Nectariferous disc; h.Conical and not uniform disc cells.Note some of them with a greater length; i-l.B. verticillata, i. Flower bud.Note the spatulate calyx lobes and the hypanthium pilose in the upper part; j.Shortly bifid stigma, showing papillae at the inner surface; k.Nectariferous disc; l.Isodiametric and uniform disc cells.

Figure 4 -
Figure 4 -Fruit and seed morphology.a-h.Fruits.a-f.Diacrodon compressus, a. Fruit and indehiscent valves; b.Cross section of a fruit with one seed (SEM); c.Cross section of a fruit with two seed (SEM); d.Cross section of fruit with one seed (MO); e. Cross section of fruit with a mature seed and with an aborted seed (MO); f.Cross section of fruit with two seeds (MO); g.Borreria spinosa, Fruit and valves, both dehiscent; h.Borreria verticillata, Fruits and valves.Note the dehiscence pattern variation of the valves; i-k.Seeds.i. Diacrodon compressus, lateral and narrowly elliptic ventral view.Note the lateral compression of the seed and the narrowly groove; j.Borreria spinosa, dorsal and elliptic to obovate ventral view.Note the wide groove; k.B. verticillata, dorsal and obovate ventral view.Note the wide groove.