Phylogeny of Muhlenbergia subg. Pseudosporobolus, including M. spatha (Poaceae, Chloridoideae, Cynodonteae, Muhlenbergiinae) now found in Zacatecas, Mexico.

Muhlenbergia spatha, previously known only from near the type locality in San Luis Potosí, is reported from two localities in Zacatecas, Mexico. Historically, botanists have overlooked this diminutive annual. To clarify affinities of M. spatha, we present a molecular phylogeny emphasising species in M. subg. Pseudosporobolus using sequence data from two plastid markers (rpl32-trnL and rps16 intron) and nrDNA ITS. In addition, we include an updated description, illustration and discussion of the habitat of M. spatha.

Introduction

Columbus is a small (usually less than 20 cm tall) annual first collected in the mountains of San Miguelito, in the valley of San Luis Potosí by J.G. Schaffner in 1876 (Bentham 1882). There are few collections of this species (only two known numbers collected by Schaffner with at least 14 duplicates) and it was thought to be extirpated until it was rediscovered near the type locality by J.T. Columbus along the Río Potosino in the Sierra de San Miguelito in 2001 (Columbus et al. 2001). can be separated from other species in the genus by its possessing small, few-branched condensed panicles that are partially included in a spatheolate sheath with each branch containing 1–5 sessile spikelets.

Bentham (1882) thought (Benth.) Nash (≡ Benth. ≡ ) was related to other members of the (e.g. Willd. and P. Beauv = Schreb.), but he also suggested the inflorescences were nearer to members of the or Pappophoreae. Beal (1896), Nash (1912), and Conzatti (1988) followed Bentham and included the monotypic Nash in the . There are no modern treatments of in Mexico, although a fairly complete genus and species description is given in Nash (1912) and, later, much abbreviated in Conzatti (1988). Pilger (1956) placed in the tribe Link, again near and . Clayton and Renvoize (1986) indicated was “an isolated genus apparently related to ” J. Presl (= Lag.; Peterson et al. 2015) and placed it in the tribe . Currently, the includes 25 subtribes, 95 genera and 859 species (Peterson et al. 2010a, 2016, 2017; Soreng et al. 2017). Molecular DNA sequence studies support placement of in the , aligning the genus within the monogeneric subtribe Pilg. in (Parodi) P.M. Peterson (Columbus et al. 2010; Peterson et al. 2010a, 2010b). There are at least 27 species of that align within .

In this paper, we report two new collections of in Zacatecas, include a complete updated description and illustration and present a molecular phylogeny of species in using sequence data from two plastid markers (rpl32-trnL and rps16 intron) and a single nuclear marker (ITS). Our new molecular phylogeny of was included to characterise evolutionary relationships of , since earlier analyses for this species were based on few markers (Columbus et al. 2010; Peterson et al. 2010b). Based on a single sequence (rpl32-trnL), paired with (Griseb.) P.M. Peterson & Columbus in a plastid tree and it paired with Vasey ex J.M. Coult. in a combined (plastid and ITS) tree (Peterson et al. 2010b).

Materials and methods

Phylogenetic analyses. Detailed methods for DNA extraction, amplification, sequencing and phylogenetic analysis are given in Peterson et al. (2010a, 2010b, 2014, 2015, 2016). In brief, we estimated the phylogeny amongst members of based on the analysis of three molecular markers (nuclear ribosomal ITS 1&2; plastid rpl32-trnL and rps16 intron DNA sequences). For this study we included a sampling of species within the five subgenera of , (Kunth) Swallen and the outgroups (Nees ex Kunth) Arechav. (, ), (L.) R. Br. (, ) and Hack. (, ) [Peterson et al. 2010b, 2016]. Voucher information and GenBank numbers for all 41 samples used in the analysis are given in Table 1.

Table 1.

Taxon voucher (collector, number and where the specimen is housed), country of origin and GenBank accession for DNA sequences of rps16 intron, rpl32-trnL and ITS regions (bold indicates new accession); a dash (–) indicates missing data.

N	Taxon	Voucher	Country	rps16 intron	rpl32-trnL	ITS
	OUTGROUP
1	Distichlis scoparia var. erinacea (Nees ex Kunth) Arechav.	Peterson 17475, Soreng & Refulio-Rodriguez (US)	Argentina, Neuquen	GU360477	GU359803	GU359334
2	Sporobolus indicus (L.) R. Br.	Peterson 22025 & Saarela (US)	Mexico, Chihuahua	GU360355	GU359913	GU359209
3	Willkommia sarmentosa Hack.	Schweickerdt 2181 (US)	South Africa,	GU360343	GU359924	GU359194
	MUHLENBERGIA
4	Muhlenbergia ramulosa (Kunth) Swallen	Peterson 22447 & Saarela (US)	Mexico, Durango	GU360406	GU359953	GU359115
	subg. Bealia
5	Muhlenbergia arenicola Buckley	Peterson 19947 & Lara-Contreras (US)	Mexico, Coahuila	GU360413	GU359960	GU359166
6	Muhlenbergia tricholepis (Torr.) Columbus	Peterson 22099 & Saarela (US)	Mexico, Chihuahua	GU360305	GU359853	GU359278
	subg. Clomena
7	Muhlenbergia durangensis Y. Herrera	Peterson 13644, Knowles, Dietrich, Braxton & Gonzalez-Elizondo (US)	Mexico, Durango	HM143552	HM143162	HM143060
8	Muhlenbergia montana (Nutt.) Hitchc.	Peterson 22234 & Saarela (US)	Mexico, Sinaloa	GU360417	GU359964	GU359162
	subg. Muhlenbergia
9	Muhlenbergia glauca (Nees) B.D. Jacks.	Peterson 21023, Saarela, Lara Contreras & Reyna Alvarez (US)	Mexico, Coahuila	HM143563	HM143173	HM143072
10	Muhlenbergia pereilema P.M. Peterson	Peterson 22191 & Saarela (US)	Mexico, Sinaloa	GU360282	GU359993	GU359131
	subg. Pseudosporobolus
11	Muhlenbergia alopecuroides (Griseb.) P.M. Peterson & Columbus	Peterson 20960, Saarela, Lara Contreras & Reyna Alvarez (US)	Mexico,	GU360426	GU359976	GU359152
12	Muhlenbergia alopecuroides (Griseb.) P.M. Peterson & Columbus	Peterson 22008 & Saarela (US)	Mexico, Chihuahua	GU360425	GU359975	GU359153
13	Muhlenbergia arenacea (Buckley) Hitchc.	Peterson 10624 & Annable (US)	Mexico, Coahuila	GU360414	GU359961	GU359165
14	Muhlenbergia asperifolia (Nees & Meyen ex Trin.) Parodi	Peterson 15452, Soreng, Finot & Judziewicz (US)	Chile, Region III (Atacama)	HM143539	HM143149	HM143048
15	Muhlenbergia atacamensis Parodi	Peterson 19626, Soreng, Salariato, & Panizza, (US)	Argentina, Jujuy	GU360489	GU359879	GU359344
16	Muhlenbergia cuspidata (Torr. ex Hook.) Rydb.	Hill 35331 (US)	USA	HM143546	HM143156	HM143055
17	Muhlenbergia decumbens Swallen	Columbus 3653 (RSA)	Mexico	–	–	EF153029
18	Muhlenbergia fastigiata (J. Presl) Henrard	Peterson 21512, Soreng, LaTorre & Rojas Fox (US)	Peru, Ancash	HM143556	HM143166	HM143064
19	Muhlenbergia implicata (Kunth) Trin.	Peterson 22266, Saarela (US)	Mexico, Oaxaca	HM143568	HM143179	HM143077
20	Muhlenbergia jaime-hintonii P.M. Peterson & Valdés-Reyna	Peterson 15841 & Valdés-Reyna (US)	Mexico, Nuevo León	HM143569	HM143181	HM143079
21	Muhlenbergia ligulata (E. Fourn.) Scribn. & Merr.	Peterson 22416 & Saarela (US)	Mexico, Durango	GU360440	GU359863	GU359273
22	Muhlenbergia monandra Alegría & Rúgolo	Peterson 17990 & Refulio-Rodriguez (US)	Peru, Lima	–	–	GQ397891
23	Muhlenbergia multiflora Columbus	Peterson 7845 & Annable (US)	USA, Colorado	GU360289	GU359985	GU359138
24	Muhlenbergia palmirensis Grignon & Lægaard	Peterson 9317 & Judziewicz (US)	Ecuador, Chimborazo	HM143586	HM143200	HM143098
25	Muhlenbergia paniculata (Nutt.) Columbus	Peterson 12070 & Annable (US)	USA, Colorado	GU360375	GU359936	GU359201
26	Muhlenbergia phleoides (Kunth) Columbus	Peterson 24452, Romaschenko & Valdés-Reyna (US)	Mexico, Nuevo León	–	MH400231	MH400228
27	Muhlenbergia phleoides (Kunth) Columbus	Peterson 24799, Romaschenko, Rodriguez Avalos, Herrera-Simoni, & Garcia Rodriguez (US)	Mexico, Aguascalientes	–	MH400232	MH400229
28	Muhlenbergia pungens Thurb. ex A. Gray	Ricketson 4642 (MO)	USA, Arizona	MH508106	MH508102	MH508098
29	Muhlenbergia repens (J. Presl) Hitchc.	Peterson 7900 & Annable (US)	USA, New Mexico	HM143596	HM143212	HM143110
30	Muhlenbergia richardsonis (Trin.) Rydb.	Peterson 7832 & Annable (US)	USA, Colorado.	HM143598	HM143214	HM143112
31	Muhlenbergia seatonii Scribn.	Peterson 9946	Mexico, Puebla	MH508107	MH508103	MH508099
32	Muhlenbergia spatha Columbus	Schaffner 134 (US)	Mexico,	–	GU359981	MH400230
33	Muhlenbergia subbiflora Hitchc.	Peterson 21158, Saarela, Rosen & Reid (US)	Mexico, Durango	GU360439	GU359877	GU359318
34	Muhlenbergia tenuissima (J. Presl) Kunth	Peterson 4751 & Annable	Mexico, Jalisco	MH508108	MH508104	MH508100
35	Muhlenbergia uniflora (Muhl.) Fernald	Peterson 13212, Annable, Pizzolato, Gordon, Frett, Frick, Morrone & Griner (US)	USA, New Jersey	HM143616	HM143232	HM143130
36	Muhlenbergia uniflora (Muhl.) Fernald	Peterson 20862 & Saarela (US)	USA, New York	GU360275	GU359994	GU359119
37	Muhlenbergia utilis (Torr.) Hitchc.	Peterson 24869 & Romaschenko (US)	Mexico, San Luis Potosí	–	MH508105	MH508101
38	Muhlenbergia villiflora Hitchc.	Peterson 15811 & Valdés-Reyna (US)	Mexico, Nuevo León	HM143620	HM143236	HM143133
39	Muhlenbergia wrightii Vasey ex J.M. Coult.	Peterson 20964, Saarela, Lara Contreras & Reyna Alvarez (US)	Mexico, Coahuila	HM143623	HM143240	HM143137
	subg. Trichochloa
40	Muhlenbergia rigens (Benth.) Hitchc.	Peterson 22129 & Saarela (US)	Mexico, Chihuahua	GU360357	GU359951	GU359117
41	Muhlenbergia longiligula Hitchc.	Peterson 15224 & Cayouette (US)	USA, Arizona	HM143574	HM143187	HM143085

Results and discussion

Phylogeny. A total of 16 new sequences from (Kunth) Columbus, Thurb. ex A. Gray, Scribn., (J. Presl) Kunth and (Torr.) Hitchc. are reported in GenBank (Table 1). Total aligned characters for individual regions and other parameters are noted in Table 2. In Figure 1, we combined the plastid–ITS sequences in our analysis since there was little incongruence between these data sets.

Table 2.

Characteristics of the three regions, rpL32-trnL, rps16 intron and ITS and parameters used in Bayesian analyses indicated by Akaike Information Criterion (AIC).

	rpL32-trnL	rps16 intron	Combined plastid data	ITS	Overall
Total aligned characters	996	1088	2084	761	2845
Likelihood score (-lnL)	4758.44	3429.94		9569.67
Number of substitution types	6	6	−	6	−
Model for amongst-site rate variation	gamma	gamma	−	gamma	−
Substitution rates
rAC	1.1071	1.2315	−	1.5168	−
rAG	1.8768	1.2968		2.8806
rAT	0.4688	0.4669		1.9244
rCG	1.2702	1.0243		0.7054
rCT	1.4748	2.4545		5.3115
rGT	1.0000	1.0000		1.0000
Character state frequencies
fA	0.3827	0.3860	−	0.2585	−
fC	0.1189	0.1105		0.1967
fG	0.1241	0.1772		0.2539
fT	0.3740	0.3261		0.2907
Proportion of invariable sites	0.1608	0.3844	−	0.3790	−
Substitution model	TVM+G	TVM+G	−	GTR+I+G	−
Gamma shape parameter (α)	0.9290	0.9303	−	0.7988	−

Figure 1.

Maximum-likelihood tree inferred from combined plastid (rpl32-trnL, rps16 intron) and ITS sequences. Thick branches indicate posterior probabilities of 1; numbers above branches are posterior probabilities less than 1 but greater than 0.50; Scale bar: 2%.

The maximum-likelihood tree from the combined analysis of ITS and two plastid regions (rpL32-trnL and rps16 intron) is well resolved, with strong support for the monophyly of , including and five subgenera, , , , and [Fig. 1; posterior probability (PP) = 1 for thick branches]. Within , is strongly supported (PP = 1) as sister to and . Swallen, Alegría & Rúgolo, , and are newly reported as occurring in , bringing the total to 27 species for this subgenus.

It is interesting but not surprising that , a close relative of , was found as an associated species at both collection sites in Zacatecas (; Peterson 25544 & Herrera Arrieta). , , and Kunth (a presumed member of this clade, not yet sampled and not included in our tree) all have plumbeous-mottled spikelets that disarticulate as a unit (below the glumes) leaving a small cuplike tip and glumes with 2–5 recurved awns (Reeder 2003; Peterson et al. in prep.). Plumbeous-mottled spikelets and well-developed sclerenchyma girders in the primary vascular bundles are additional traits shared by members of (Watson and Dallwitz 1992; Peterson and Herrera Arrieta 2001; Peterson et al. 2010b).

Taxonomy

Columbus, Aliso 28: 66. 2010, non Muhlenbergia gracilis (Kunth) Trin. (1824).

Fig. 2A–D

Figure 2.

: A Habit B Panicle partially enclosed by the leaf sheath C Basal panicle branch with a lower sterile floret and small cuplike structures (callus remains) where the fertile spikelets were inserted D Spikelet, side view E Upper glume showing three recurved awns, dorsal view. (Peterson 25544 & Herrera Arrieta, CIIDIR).

Benth., Hooker’s Icon. Pl. 14: 59, t. 1378. 1882.

Description.

Delicate annuals, loosely caespitose. Culms 5–20(–30) cm tall, erect, geniculate below; nodes scaberulous, branching at lower and middle nodes; internodes 2.0–4.5(–8.0) cm long, strongly 4–6 ribbed. Leaves caulescent and basal; ligules 0.8–1.8 mm long, membranous, decurrent, apex obtuse, minutely erose; sheaths 0.8–1.5 cm long, much shorter than the internodes, oblong, open, chartaceous, strongly ribbed with 7–9-veins, sometimes keeled, margins hyaline; blades 0.5–4 cm long, 0.5–1.5 mm wide, flat or folded, adaxially scattered pubescent near base, the hairs antrorse leaning, apex obtuse. Inflorescences compound, fasciculate, composed of terminal and axillary condensed panicles, these branched near the base, the basal-most branch usually with a sterile floret at the base consisting of two scales, the entire panicle partially included in a spatheolate sheath; sterile florets 2–4 mm long, 0.8–1 mm wide, linear-apiculate, flat, hyaline, 1-veined; racemose branches each bearing 1–5 fertile sessile spikelets, the spikelets separated by 1–4 mm on each branch; rachis angular, 3 or 4-ribbed. Spikelets 4.4–6 mm long, lanceolate, laterally compressed, solitary, composed of one fertile floret, plumbeous-mottled; rachilla not extended; callus short, blunt, pubescent, located just below the glumes where disarticulation occurs leaving a small cuplike tip; glumes dimorphic; lower glumes absent or obscure; upper glumes 3.5–5(–6) mm long, about as long as the lemma, oblong, chartaceous, firmer than fertile lemma (excluding the awns), 7–9-veined, lateral veins ribbed, pubescent along the veins on lower ½, apex deeply bifid and 3(5)–awned, the awns 5–7 mm long, scabrous, recurved, arising between the bifid apex; lemmas (4–)4.8–6 mm long, lanceolate, membranous, 3-veined, keeled, midvein scaberulous, lateral veins ribbed, apex acute to acuminate, minutely bifid, 1-awned, the awn 3–5 mm long arising from between the teeth; paleas 3.7–5.5 mm long, shorter than the lemmas, oblong, hyaline to membranous, tightly involute, 2-veined, apex obtuse, unawned; stamens 3; anthers 2–2.5 mm long, yellowish; ovary glabrous; caryopses 1.8–2 mm long, 0.5 mm wide, narrowly fusiform, straw coloured.

Distribution.

The species is known only from the type locality in San Luis Potosí and Municipio Villanueva, Zacatecas.

Ecology.

was found by the authors growing on flat table rock in open areas near arroyos associated with Lag., (Michx.) Torr., (Retz.) Alston, , , (Kunth) Trin., , (Kunth) Kunth, Kunth, (A. Rich.) Stapf, Desv., L., Humb. & Bonpl. ex Willd., P. Beauv., (Kunth) Hitchc., Hitchc., (Michx.) Nees and Hochst. ex A. Rich.

Habitat.

At the southern edge of the Sierra Madre Occidental Range in the Sierra Fría de Aguascalientes, we recently found in two localities south of the city of Zacatecas in a corridor located east of Villanueva: 1) 17.4 km east of Villanueva on flat table rock just above Arroyo “El Muerto” in open areas and 2) about 1 km northeast of the small village Palomas Viejas along Arroyo Juan Manuel on natural grasslands near cultivated fields. These two sites are approximately 204 km W (air distance) from the type locality southwest of San Luis Potosí in the Sierra de San Miguelito along the Río Potosino (Columbus et al. 2001). According to Instituto Nacional de Estadística, Geografía e Informática [INEGI] (2003, 2005), the Sierra San Miguelito is placed in the Mesa del Centro Province in the Sierra Madre Oriental. Near Villanueva, Zacatecas, the soils are Haplic Phaeozem (forming sodium carbonate, Na2CO3) over extrusive igneous rock (acidic) in a semi-dry climate with an annual mean temperature of 16°C and an annual mean precipitation of 60 cm (INEGI 2008a, 2008b). In the Sierra de San Miguelito along the Rio Potosino, the soils are Calcic Regosol (forming calcium carbonate, CaCO3) of medium texture over extrusive igneous rock (acidic) in a semi-dry climate with an annual mean temperature of 16 °C and an annual mean precipitation of 40 cm (INEGI 2008a, 2008b). Without field verification, it is uncertain whether the soil differences among these sites are significant, but we are reasonably certain that all are alkaline with an elevated pH.

Conservation status.

The species is rare in Mexico and is known from only three recent collections. Since it is a diminutive, short-lived annual, the species is easily overlooked and the main concern seems to be loss of habitat via human impact, i.e. agriculture, dam and road construction.

Specimens examined.

Mexico. San Luis Potosí: 1876, J.G. Schaffner 134 (GOET-006918 [image!], US-825687); Sierra de San Miguelito, Río Potosino, , 1980 m, 2 Oct 2001, J.T. Columbus 4040 (RSA [image! in Columbus et al. 2001]). Zacatecas: Mpio. Villanueva, Arroyo Juan Manuel ± 1 km NE of Palomas Viejas near Villanueva, , 2112 m, 22 Oct 2006, O. (CIIDIR); Mpio Villanueva, 10.8 mi [17.4 km] E of Villanueva, just above arroyo “El Muerto”, , 2083 m, 2 Oct 2015, P.M. Peterson 25544 & Y. Herrera Arrieta (CIIDIR, US).

Comments.

The hand written script (verified by J. Rzedowski, per. comm., also see Rzedowski 1959) on the label of the holotype (K) says: “Müehlenbergia gracilis mihi”, indicating that Jose Guillermo Schaffner thought he had collected a new species of . Bentham (1882) agreed with Schaffner that it was a new species but thought it had enough unique morphological features (spatheolate sheathed panicles, sessile spikelets and 3(5)-awned upper glumes) to warrant description of a new genus.