Plant Systematics and Evolution (2021) 307:34 https://doi.org/10.1007/s00606-021-01756-1

ORIGINAL ARTICLE

Cytogenetics of wild of the family ()

Pedro Jara‑Seguel1,2 · Paola Jara‑Arancio3,4 · Elías Andrade2 · Jonathan Urrutia‑Estrada5 · Claudio Palma‑Rojas6 · Cristian Araya‑Jaime7

Received: 16 October 2020 / Accepted: 19 March 2021 © The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2021

Abstract Studies on cytogenetic diversity within the Alstroemeriaceae family along its intercontinental distribution are scarce when comparing species of their four genera. This study aimed at exploring the cytogenetic diversity of the family along their geographical distribution, including species that inhabit Oceania and . Using quantitative and qualitative methods, we analysed 50 species of the four genera (24% of the species of the family), comparing cytogenetic characters such as chromosome number, karyotype morphology and asymmetry, location of chromosome satellites, total haploid length, meiotic behaviour, C and FISH banding, and DNA C-values. We concur with other authors in that generic delimitation based on chromosome number is clear among (2n = 16) and (2n = 18), both belonging to Alstroemerioideae. Moreover, these two genera have diferent chromosome numbers compared to those of and (both 2n = 20) belonging to Luzuriagoideae. The above observations were also supported by the phylogeny reconstructed here. In addition, analyses on karyotype morphology and asymmetry have determined gradual diferences in nuclear architecture among genera and species. We concluded that the cytogenetic diversity described within Alstroemeriaceae is concentrated mainly in South America, which is to be expected given the higher species diversity that has been described for that continent. Although the presence of three chromosome numbers within Alstroemeriaceae has no relation with their wide geographical distribution in two continents, an appreciable variation was observed in karyotype morphology and asymmetry, total haploid length and cytomolecular features when the species were compared along their geographical range.

Keywords Alstroemerioideae · Asymmetry · Chromosome number · Karyotype morphology · Luzuriagoideae

Introduction

Handling editor: Martin A. Lysak. The Alstroemeriaceae family includes two subfamilies with * Paola Jara‑Arancio four genera and 204 species distributed in Gondwanean [email protected] regions along , South America, and Oce- 1 Departamento de Ciencias Biológicas y Químicas, ania. The current evidence shows that the main centre of Universidad Católica de Temuco, Temuco, Chile genera diversity is located in southern Chile, where taxa of 2 Núcleo de Estudios Ambientales, Facultad de Recursos three genera of the family representing the two subfamilies Naturales, Universidad Católica de Temuco, Temuco, Chile are present, thus comprising ca. 13 species in that region 3 Instituto de Ecología y Biodiversidad, Universidad de Chile, (Bayer 1987; Rodríguez and Marticorena 1987; Muñoz- Santiago, Chile Schick and Moreira-Muñoz 2003). From a biological point 4 Departamento de Ciencias Biológicas, Departamento de of view, the species display various life forms, some as vines Ecología y Biodiversidad, Facultad de Ciencias de la Vida, living in the , others erect, inhabiting the fringes of Universidad Andrés Bello, Santiago, Chile forests or growing in open prairies (Bayer 1987; Rodríguez 5 Laboratorio de Invasiones Biológicas, Facultad de Ciencias and Marticorena 1987; Arroyo-Leuenberger 1988a, b; Forestales, Universidad de Concepción, Concepción, Chile Arroyo-Leuenberger 2001). These are characterised 6 Departamento de Biología, Universidad de La Serena, by striking fowers in the case of the Alstroemerioideae sub- La Serena, Chile family, and attractive fronds and in the case of the 7 Instituto de Investigación Multidisciplinar en Ciencia y Luzuriagoideae subfamily. For this reason Alstroemeriaceae Tecnología, Universidad de La Serena, La Serena, Chile

Vol.:(0123456789)1 3 34 Page 2 of 22 P. Jara-Seguel et al. species have a great ornamental attraction as cut fowers or contrasts with that described for other families within the potted and garden plants (Arroyo and Leuenberger 1988b; Liliales such as , Colchicaceae, and Melanthiaceae. Muñoz-Schick and Moreira-Muñoz 2003). For these three families, cytogenetic characters (e.g., karyo- The frst report on the cytogenetics of Alstroemeriaceae types, chromosome number, ploidy, asymmetry, and genome was published by Strasburger (1882) in Alstroemeria chil- size) have been focused on interpreting evolutionary trends, ensis Lem. (≡ L.). Later on, various by superimposing onto phylogenetic reconstructions (Sato works focused on studying natural populations and culti- 1943; Tamura 1995; Peruzzi et al. 2009; Chacón et al. 2014; vars of Alstroemeria and Bomarea (Taylor 1926; Whyte Pellicer et al. 2014), thus giving consistency to their current 1929; Sato 1938; Darlington and Wylie 1955; Cave 1967; biosystematic framework. Broertjes and Verboom 1974; Hughes 1987; Tsuchiya and In this work, we analysed cytogenetic diversity within Hang 1987, 1989; Hang and Tsuchiya 1988; Hunziker and Alstroemeriaceae on the basis of the studies available for 50 Xifreda 1990; Rustanius et al. 1991). These works provided wild species inhabiting a wide geographical range in South a methodological basis for the studies published in subse- America and Oceania. We query whether this extensive quent years, and have revealed interesting characteristics of latitudinal and longitudinal range correlates with the high the chromosomes such as their large size, shape, and satellite cytogenetic diversity within the family, analysing multiple location. During the last decades, studies on the cytogenet- characters and seeking consistency with the morphologi- ics of Alstroemeriaceae have increased, totalling 43 articles cal and molecular variations previously described for the between 1929 and 2020, which have described the chromo- species (Bayer 1987; Han et al. 2000; Hofreiter and Tillich some numbers and karyotype morphology of mitotic and 2002; Muñoz-Schick and Moreira-Muñoz 2003; Hofreiter meiotic chromosomes. Other articles have documented the and Rodríguez 2006; Hofreiter 2007; Chacón et al. 2012a, application of specifc banding methods (C and FISH band- b; Conran et al. 2014). Based on the variety of cytogenetic ing; Buitendijk and Ramanna 1996; Jara-Seguel et al. 2004, studies available so far for the family, with 24% of the spe- 2005; Baeza et al. 2007a; Kamstra et al. 1999, 2004; Zhou cies studied throughout its wide geographical range, our et al. 2003; Chacón et al. 2012a), and 2C-value estimations hope is that it will be possible to observe high cytogenetic (Buitendijk et al. 1997, 1998). diversity in their genera and species, particularly in South At present, cytogenetic descriptions have been accu- America where the largest number of taxa live and have been mulated for Alstroemeriaceae, analysing single species studied cytologically. Several quantitative methods are used or groups of them. However, few studies have focused here to answer our queries and test the hypothesis, and dif- on analysing cytogenetic diversity within the family on a ferent previously reported cytological markers are analysed geographical scale or on the basis of climatic variation, to describe the variation. including comprehensive dataset gathering. Pioneering approaches in this regard are the works reported by Bui- tendijk and Ramanna (1996) and Buitendijk et al. (1997), Material and methods who studied species of the Alstroemeria inhabiting arid climates as well as humid and cold climates, giving Source of cytogenetic data rise to discussions about interesting tendencies on genome complexity and dynamics based on C-banding pattern and Cytogenetic data of wild Alstroemeriaceae species were genome size. Moreover, advances in the use of molecular compiled from 43 articles published to date between 1929 tools (AFLP and RAPD markers) studying genetic diver- and 2020. A data matrix (Table 1) was assembled includ- sity within the family were reported by Han et al. (2000), ing features such as chromosome number (somatic 2n, and Baeza et al. (2007a), Finot et al. (2018). Other eforts have meiotic n), haploid karyotype formula, total haploid length focused on studying the chromosome distribution of rDNA (THL), asymmetry (MCA and CVCL indices), and C-value at interspecifc levels within Alstroemeria (Baeza et al. estimations. Chromosome details such as satellite location 2007a; Chacón et al. 2012a; Finot et al. 2018) as well as are shown in Table 3, meiotic analysis in Table 4, C-banding the biogeography of the family interpreted in a phylogenetic pattern in Table 5, and molecular cytogenetics (FISH, GISH, context (Vinnersten and Bremer 2001; Chacón et al. 2012b; and other markers) are shown in Table 6. Geographical loca- Givnish et al. 2016; Finot et al. 2018). All these studies tion was provided including countries and geo-references have provided a basis to hypothesize on the cytogenetic and according to original sources. For those species in which molecular variation that may be present within the family collected sites were registered in the original source but along its wide geographic distribution, although attention not geo-referenced in geographic coordinates, the specifc has centred mainly on Alstroemeria and scant interest has location was looked up on Google Earth Pro and processed been shown in Bomarea, Luzuriaga, and Drymophila. The in kml format. Later, the kml file was processed using paucity of studies regarding these Alstroemeriaceae genera the ArcGis 10.4 software and then processed with a kml

1 3 Cytogenetics of Alstroemeriaceae Page 3 of 22 34

Australia; Tas ; ( 1996 ) ( 1996 ) ( 1996 ) Baeza et al. ( 2010a , b ) Baeza et al. Baeza et al. ( 2010a , b ) Baeza et al. Buitendijk et al. ( 1997 ) et al. Buitendijk Baeza et al. (2009) Baeza et al. Baeza et al. (2009) Baeza et al. Sanso ( 2002 ) Baeza et al. ( 2010a , b ) Baeza et al. Meerow et al. ( 1999 ) et al. Meerow Jara-Seguel et al. ( 2004 ) et al. Jara-Seguel Baeza et al. (2016b) Baeza et al. Baeza et al. ( 2010a , b ) Baeza et al. Chacón et al. ( 2012a ) Chacón et al. Buitendijk et al. ( 1997 ) et al. Buitendijk Buitendijk et al. ( 1997 ) et al. Buitendijk Buitendijk et al. ( 1997 ) et al. Buitendijk Buitendijk et al. ( 1997 ) et al. Buitendijk Buitendijk and Ramanna and Ramanna Buitendijk Buitendijk and Ramanna and Ramanna Buitendijk Sanso ( 2002 ) References Buitendijk and Ramanna and Ramanna Buitendijk Sanso ( 2002 ) 52 55.5 50.2 55.3 50.5 50.8 2C-value (pg) 2C-value ; Aus Islands; Nze New Falkland 21.3–8.8 22.5–7.5 27.1–9.2 20.2–7.8 21.0–8.7 16.4–3.4 19.2–5.0 16.0–4.2 25.0–13.0 24.4–8.8 22.8–5.6 16.2–5.4 L–S (µm) - t + 3 t + 2 t + 1 t + 3 t + 3 t + 1 t + 1 t + 1 st + 2 t + 1 t (3B) 4 m + 2 st 3 m + 1 sm st 3 m + 1 sm 4 st 2 m + sm st 8 bivalents 4 m + 2 st 1 m + 2 sm 4 st 3 m + 1 sm st 2 m + sm 1 st 3 m + 1 sm 4 st 4 m + 3 st 3 m + 1 sm t st 2 m + 1 sm 4 st karyotype formula ( n) formula karyotype 8 bivalents, (1 or 2 B) 8 bivalents, 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 2 n ; Col Colombia; Fis Ecu Ecuador; 89.6 78.5 90.4 92.0 53.9 62.0 59.8 76.0 78.6 78.0 125.5 THL (µm) Brazil; Per Brazil; 37 43 40 48 38 53 45 47 41 41 47 48 MCA 35 64 35 35 32 64 48 50 33 32 54 50 CVCL Chi Chi Chi Chi Chi Chi Chi Bra Chi Chi Chi Bra Bra Bra Chi Chi Chi Arg Bra Bra GD Chi ; Chi Chile; Bra Argentina; 71° 32 ′ W 71° 29 ′ W 72° 25 ′ W 73° 09 ′ W 71° 37 ′ W 71° 31 ′ W 47° 85 ′ W 70° 29 ′ W 71° 29 ′ W 71° 43 ′ W 49° 5 ′ W 52° 58 ′ W Longitude 69° 25 ′ W 52° 43 ′ W 52° 43 ′ W 71° 37 ′ W 32° 56 ′ S 31° 53 ′ S 35°–37°S 36° 45 ′ S 36° 46 ′ S 30° 55 ′ S 31° 42 ′ S 20° 39 ′ S 25° 24 ′ S 32° 02 ′ S 33° 05 ′ S MBG 24° 17 ′ S 8° 18 ′ S 36°–47°S 36°–47°S 33°S Latitude 31° 20 ′ S 8° 18 ′ S 8° 18 ′ S Localities 30° 55 ′ S Ahr Ahm Ahv Ahc Ahg Agr Add Adc Aau Aag Aav Ain Code Cytogenetic features and collection sites of the Alstroemeriaceae species studied. MBG, Missouri Botanical Garden. Code, for species included in asymmetry analysis only; 2C-value, 2C-value, only; species included in asymmetry and collection sites of the analysis MBG, Missouri Alstroemeriaceae species studied. Code, for Botanical Garden. features Cytogenetic cf. rupestris M.C. Assis (Herb.) Ehr.Bayer Ehr.Bayer hookeri giana ana (Herb.) Ehr.Bayer bol. & F.K.Mey Ehr . Bayer Ehr.Bayer angustifolia (Phil.) Muñoz- venustula Schick A. hookeri subsp. r ecumbens A. hookeri subsp. maculata A. hookeri subsp. hookeri A. hookeri subsp. hookeri A. subsp. hookeri Sweet A. hookeri subsp. cummin - A. hookeri subsp. cummingi - - Tom Meerow, A. hygrophila Phil A. graminea A. subsp. diluta diluta A. subsp. chrysantha diluta A. A. caryophyllaea Jacq Spreng A. brasiliensis A. aurea Graham A. aurea Herb. subsp. A.angustifolia Alstroemeria andina var. Alstroemeria A. inodora Herb A. inodora 1 Table geographical distribution; content. GD geographical Argnuclear DNA S length pair pair; shortest chromosome chromosome L length longest Tasmania; Taxon A. hookeri subsp. r ecumbens

1 3 34 Page 4 of 22 P. Jara-Seguel et al. ( 1996 ) ( 1996 ) ( 1996 ) Buitendijk et al. ( 1997 ) et al. Buitendijk Sanso ( 2002 ) Baeza et al. ( 2011a , b ) Baeza et al. Sanso ( 2002 ) Negritto ( 2015 ) et al. Baeza et al. ( 2018a ) Baeza et al. Baeza et al. ( 2018a ) Baeza et al. Buitendijk et al. ( 1997 ) et al. Buitendijk Baeza et al. ( 2018a ) Baeza et al. Buitendijk and Ramanna and Ramanna Buitendijk Baeza et al. ( 2018a ) Baeza et al. Baeza et al. ( 2006 ) Baeza et al. Buitendijk et al. ( 1998 ) et al. Buitendijk Baeza et al. ( 2006 ) Baeza et al. Buitendijk et al. ( 1997 ) et al. Buitendijk Buitendijk and Ramanna and Ramanna Buitendijk Buitendijk et al. ( 1998 ) et al. Buitendijk Sanso and Hunziker ( 1998 ) Sanso and Hunziker Sanso ( 2002 ) Sanso and Hunziker ( 1998 ) Sanso and Hunziker References Baeza al. (2008) Baeza al. (2008) Buitendijk and Ramanna and Ramanna Buitendijk Baeza et al. ( 2020 ) Baeza et al. 44.0 41.9 68.5 69.4 69.4 70.0 2C-value (pg) 2C-value 12.0–4.0 21.8–8.2 15.6–3.8 27.5–6.7 20.6–4.4 17.0–3.5 19.6–11.9 20.3–11.4 15.0–8.0 24.8–6.8 24.8–6.1 20.0–7.8 21.7–6.8 21.3–4.7 L–S (µm) + 2 t − − sat + 1 + 4 t + 1 t + 1 st + 1 t + 1 t + 2 t + 2 t + 4 st + 1 st , 1 t t + 1 − sat sat + 2 t 8 bivalents 1 m + 3 sm st 8 bivalents 2 m + sm 1 st , 1st, 4 t 2 m , 1 sm 1st, 2 m + 1 sm 4 st 2 m , 1 sm 4 st 2 m + sm − st 2 m + sm 3 st 5 m + 3 t 4 m + 1 sm 3 t 5 m + 1 sm 2 st 2 m + 1 sm − st 2 m + 1s 1 sm- − st 8 bivalents 3 m + 1 sm 4 t 4 m + 1 sm st 3 m + 1 sm 4 st karyotype formula ( n) formula karyotype 3 m + 1 sm 2 st 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 2 n 64.5 73.7 86.9 96.7 68.2 58 50.0 90.0 91.5 87.0 89.2 99.2 65.6 87.8 THL (µm) 55 55 50 42 40 51 39 39 27 52 45 48 36 46 MCA 32 55 47 60 55 55 19 21 21 50 58 37 39 52.2 CVCL Chi Chi Arg Chi Chi Chi Chi Chi Chi Chi Chi Chi Chi Chi Chi Chi Chi Arg Chi Arg Arg Chi Chi Chi GD 71° 29 ′ W 70° 21 ′ W 71° 33 ′ W 70° 16 ′ W 71° 32 ′ W 71° 14 ′ W 71° 17 ′ W 71° 19 ′ W 71° 29 ′ W 71° 24 ′ W 73° 11 ′ W 69° 25 ′ W Longitude 55° 54 ′ W 70° 22 ′ W 73° 08 ′ W 71° 24 ′ W 32° 14 ′ S 32°–3°S 39° 03 ′ S 45° 16 ′ S 33° 36 ′ S 33° 04 ′ S 29° 26 ′ S 29° 38 ′ S 29°–32°S 29° 44 ′ S 29°–32°S 31° 57 ′ S 33° 02 ′ S 36° 46 ′ S 33°–38°S 33°–38°S 34° 28 ′ S 36° 47 ′ S Latitude 27° 53 ′ S 37° 14 ′ S 37° 49 ′ S 36° 54 ′ S 32°–33°S Localities Apa Apa Amc Amt Ams Ama Amm Als Ali All Ais Apu App Ape Code (continued) C.M.Baeza Muñoz-Schick (Muñoz) Muñoz-Schick magnifca (Ehr.Bayer) Muñoz-Schick (Ehr.Bayer) gel) Ehr.Bayer gel) Bayer Bayer Ehr.Bayer presliana A. pelegrina A. L pelegrina A. patagonica A. Phil patagonica A. pallida Graham A. Negritto & marticorenae tofoensis A. magnifca var. sierrae A. magnifca var. A. magnifca subsp. A. magnifca subsp. A. magnifca Herb. subsp. magenta A. magnifca var. A. ligtu subsp. simsii A.- ligtu subsp. simsii (Spren A. ligtu subsp. A. ligtu subsp. A. ligtu L . subsp. l igtu A. Lehm psittacina A. ligtu subsp. incarnata Ehr. A. isabellana Herb A. Ehr. pseudospathulata subsp. australis A. presliana Kunth subsp. Kunth A. presliana A. pelegrina 1 Table Taxon

1 3 Cytogenetics of Alstroemeriaceae Page 5 of 22 34 ( 1996 ) Baeza et al. ( 2008a , b ) Baeza et al. Whyte ( 1929 ) Whyte Diers ( 1961 ) Baeza et al. ( 2012 ) Baeza et al. Palma-Rojas et al. ( 2007 ) et al. Palma-Rojas Jara-Seguel et al. ( 2005 ) et al. Jara-Seguel Sanso and Seo ( 2005 ) Palma-Rojas et al. ( 2007 ) et al. Palma-Rojas Hunziker and Xifreda ( 1990 ) and Xifreda Hunziker Sanso and Hunziker ( 1998 ) Sanso and Hunziker Whyte ( 1929 ) Whyte Whyte ( 1929 ) Whyte Sanso and Hunziker ( 1998 ) Sanso and Hunziker Tsuchiya and Hang ( 1989 ) Tsuchiya Baeza et al. ( 2011a , b ) Baeza et al. Meerow et al. ( 1999 ) et al. Meerow Sanso ( 2002 ) Baeza et al. ( 2018b ) Baeza et al. Baeza et al. ( 2018b ) Baeza et al. Buitendijk et al. ( 1997 ) et al. Buitendijk Tsuchiya and Hang ( 1989 ) Tsuchiya Buitendijk and Ramanna and Ramanna Buitendijk Buitendijk et al. ( 1997 ) et al. Buitendijk Guerra ( 1986 ) References Baeza and Silva ( 2016 ) Baeza and Silva Palma-Rojas et al. ( 2007 ) et al. Palma-Rojas 40.1 49.8 2C-value (pg) 2C-value 9.9–5.8 8.5–4.1 6.9–3.0 10.5–5.1 10.0–6.8 10.0–5.2 11.9–6.7 12.7–7.1 25.7–7.9 28.7–8.5 L–S (µm) − − sat + + 4 t + 2 st + 1 t + 3 t + 3 t + 1 t + 2 st + 1 t − t + 3 st + 2 st t + 1 1 t − sat 2 m + 1 sm 5 st 2 m + 1 sm 3 st 2 m + 1 sm 3 st 2 m + 3 sm st 2 m + 1 sm − st 2 m + 5 st 1 m + 3 sm st 2 m + 1 sm − st Pair 1m, heteromorphic Pair Pair 1m, heteromorphic Pair 2 m + 1 sm 3 st 2 m + 1 sm 5 st karyotype formula ( n) formula karyotype 18 18 18 18 18 18 18 18 36 18 18 18 18 16 16 16 16 16 16 16 16 16 16 18 18 18 2 n 61.7 75.4 73.1 66.8 84.7 82.5 64.5 54.4 42.5 112.0 THL (µm) 57 54 58 51 55 52 43 38 49 52 MCA 27 14 17 21 22 23 45 47 25.6 27 CVCL Col Ecu Per Chi Chi Chi Arg Chi Arg Arg Col Per Arg Chi Chi Bra Arg Chi Chi Chi Bra Bra Bra Bra Chi Chi GD 74° 22´W 78° 40´W 71° 55´W 71° 06´W 71° 07 ′ W 71° 07 ′ W 65° 75´W 69°28´W 64° 48´W 74° 27 ′ W 70° 14 ′ W 65° 28 ′ W 72° 42 ′ W 48° 97 ′ W 65° 42 ′ W 71° 25 ′ W 71° 23 ′ W 61° 50 ′ W Longitude 62° 16 ′ W 73° 04 ′ W 71° 05 ′ W 4° 24´ N 0° 28´S 13° 40´S 28° 07´S; 28° 08 ′ S 28° 08 ′ S 26° 78´S 17° 35´S 23° 50´S 4° 22 ′ N 13° 39 ′ S 23° 51 ′ S 20°–28°S 37° 48 ′ S 15° 81 ′ S 26° 21 ′ S 30° 04 ′ S 35° 10 ′ S 32°–34°S MBG 1° 12 ′ S Latitude 1° 22 ′ S 36°51 ′ S 32° 57 ′ S Localities Bsa Bpa Boi Bov Bin Bed Bbo Ave Apy Bsl Code (continued) Phil.) ovallei Leontochir (Tussac) Herb.) (Tussac) Tombol. & F.K.Mey Tombol. pulchra cea Ehr.Bayer B. edulis Herb.) B. salsilla Mirb Baker subsp. patinii B. patinii Baker Herb B. patacocencis Mirb B. ovata B. ovallei B. ovallei (Phil.) Ravenna ( ≡ (Phil.) Ravenna B. ovallei Pax B. macrocephala Baker B. involucrosa B. hirtella Herb. ( ≡ B. edulis (Tuss.) Herb B. edulis (Tuss.) B. carderi B. caldasiana Herb Bomarea boliviensis Baker Bomarea A. violacea Phil Ruiz & Pav Ruiz A. versicolor Meerow, Meerow, A. orchidioides Herb A. pygmaea Sims. subsp . A. pulchra - subsp. lavandula A. pulchra Sims A. pulchra L.f A. pulchella A. psittacina A. psittacina M.Roem. (Syn. B. salsilloides M.Roem. B. salsilla 1 Table Taxon

1 3 34 Page 6 of 22 P. Jara-Seguel et al. Jara-Seguel et al. ( 2010 ) et al. Jara-Seguel Jara-Seguel and Zúñiga ( 2005 ) and Zúñiga Jara-Seguel Jara-Seguel and Zúñiga ( 2005 ) and Zúñiga Jara-Seguel Jara-Seguel et al. ( 2010 ) et al. Jara-Seguel Beuzenberg and Hair ( 1963 ) Beuzenberg Jara-Seguel et al. ( 2010 ) et al. Jara-Seguel Moore ( 1968 ) Moore Löve ( 1985 ) Löve Wiltshire and Jackson ( 2003 ) and Jackson Wiltshire Manadue and Orchard ( 1985 ) Manadue and Orchard Sanso and Seo ( 2005 ) Hunziker and Xifreda ( 1990 ) and Xifreda Hunziker References 2C-value (pg) 2C-value 10.9–3.2 L–S (µm) 3 m + 6 sm 1 st 4 m + 3 sm st 3 m + 6 sm 1 st karyotype formula ( n) formula karyotype 20 20 20 20 20 20 20 20 20 24 18 18 2 n 48.7 THL (µm) 40 36 31 MCA 48.2 52 54 CVCL Chi Chi Chi Nze Nze Fis Fis Aus Tas Aus Arg Col GD 71° 44´W 71° 44´W 72° 18´W 175°48 ′ E 175°48 ′ E 57°50 ′ W 57°50 ′ W 147° E 147° E 151° E 65° 04´W Longitude 75° 91 ′ W 38° 27´S 38° 27´S 42° 44´S 38°58 ′ S 38°58 ′ S 51° 41 ′ S 51° 41 ′ S 36° 45´S 42° S 33° 61´S 22° 24´S Latitude 2°N Localities Lra Lpa Lma Code (continued) J.F.Macbr Enargea \\ ( ≡ Enargea & Hook.f. Gaertn.) marginata L. radicans Ruiz & Pav Ruiz L. radicans (Hook.) (Hook.) L. polyphylla L. parvifora L. parvifora Kunth L. marginata Luzuriaga Benth. Luzuriaga marginata Baker D. moorei D. cyanocarpa Drymophila R.Br Kraenzl B. stans Herb B. setacea 1 Table Taxon

1 3 Cytogenetics of Alstroemeriaceae Page 7 of 22 34 conversion tool. The fnal cartographic edition of the spe- combined Bayesian inference analysis, fve partitions were cies was mapped using the WGS 84 datum with time zone used corresponding to each gene, and the following evolu- 18 South, processed in shape format. tionary models were applied: GTR + G in ndhF; GTR + G in matK, GTR + I + G in matR, GTR + I + G in rbcL and Karyotype morphology, total haploid length, GTR + G in ITS. The Tracer programme (v1.6, Rambaut and asymmetry et al. 2003–2013) was used to visualise output parameters in order to prove stationarity and establish whether or not Diferent quantitative and qualitative parameters used to there were duplicate runs converging on the same mean describe karyotype morphology in Alstroemeriaceae are probability. Runs appeared stationary prior to ­207 genera- summarised in a data matrix (Table 1). The relative length tions; therefore we conservatively excluded the frst 2.0 × ­106 of the short arms (SA) and long arms (LA) has been useful generations of each run as burn-in. The efective sample size to describe chromosome morphology via calculation of the (ESS) value was greater than 200 in a range between 20,323 centromeric index (CI) proposed by Levan et al. (1964). SA and 32,086. Nodes with ≥ 0.95 credibility were considered and LA are also used to estimate karyotype asymmetry. An to be supported for posterior probabilities (Ronquist and additional parameter included in the data matrix was the Huelsenbeck 2003). total haploid length (THL) in micrometres for each species, as available in the literature. Diferent indices to determine karyotype asymmetry in Results Alstroemeriaceae have been provided by various authors. Here we re-estimated karyotype asymmetry for all species Representation of cytogenetic studies following the premises of Peruzzi and Eroglu (2013) in determining the Mean Centromeric Asymmetry, which was The cytogenetic analyses presented in this work are based calculated as MCA = A × 100, where A = [mean (LA − SA)/ on articles reported by the authors summarised in the data (LA + SA)], and the Coefcient of Variation of the chromo- matrix (Table 1) and other additional reports describing spe- some length calculated as ­CVCL = (Standard deviation of cifc methods cited in Tables 3, 4 and 5. Cytogenetic data chromosome length/mean of the chromosome length) × 100. were compiled for 50 wild species of Alstroemeriaceae, of It should be mentioned that these authors discussed the sta- which 31 species belong to Alstroemeria, 13 to Bomarea, tistical bases supporting the use of MCA and CVCL as the four to Luzuriaga, and two to Drymophila (24% of the spe- most adequate parameters to determine karyotype asymme- cies of the family). In total, cytogenetic data for 62 taxa try. To calculate these parameters, the relative length values were analysed, including subspecies or varieties. A detailed of the short arms (SA) and long arms (LA) of all species analysis of various cytogenetic characters evaluated for each included in the data matrix were used. In the case of those genus showed that 36% of Alstroemeria species have been works describing only chromosome number and in which a studied, 10.8% of Bomarea, 75% of Luzuriaga, but only single metaphase was shown, we frst measured the length chromosome counts have been reported for Drymophila and values of the short arms and long arms of each chromosome Luzuriaga polyphylla. pair and then calculated their respective CI, allowing us to determine their chromosome morphology. In addition, we Phylogenetic reconstructions calculated the same karyotype parameters mentioned above as representative of those species (MCA and CVCL). The phylogenetic matrix contained 5925 nucleotide char- Due to the scant data available, we estimated the cor- acters (1971 ndhF, 1262 matK, 512 matR, 1278 rbcL and relation between THL and DNA C-values, DNA C-values 902 ITS). The topology revealed a primary division between and CVCL, and DNA C-values and MCA using data for the the Luzuriagoideae (Luzuriaga/Drymophila) and Alstroeme- seven Alstroemeria species that have been studied so far. rioideae (Alstroemeria/Bomarea) subfamilies. It was noted that Bomarea and Alstroemeria are sister groups (Fig. 1). Phylogenetic analysis Geographical distribution A combined analysis for sequences of the ITS nuclear gene was conducted using four chloroplast genes: ndhF, rbcL, The 50 species (62 taxa) included in this work cover a matR and matK. Sequences for all species were downloaded wide latitudinal range from 2°N to 52°S (ca. 5500 km), from GenBank (Table 2); Smilax aspera L. (Liliales–Smi- as well as a wide longitudinal range from 47°W to 175°E lacaceae) was used as the out-group (for details see Online (Fig. 2) in two continents. On this wide distributional Resource 1), and Bayesian inference analyses were per- range, the species inhabit diverse environments including formed using MrBayes 3.2 (Ronquist et al. 2012). For the dry, humid, cold and rainy, and grow in a wide variety of

1 3 34 Page 8 of 22 P. Jara-Seguel et al.

Table 2 GenBank accession Taxon GenBank No numbers of taxa cpDNA rDNA ndhF rbcL matK matR ITS

Alstroemeria A. andina var. venustula JQ404508 JQ404769 JQ404893 JQ405004 A. angustifolia subsp. angustifolia JQ404509 JQ404770 JQ404894 A. aurea JQ404511 AY120359 JQ404771 JQ404895 JQ405005 A. caryophyllaea JQ404665 JQ404897 JQ405008 A. cf. rupestris JX418004 JX418006 JX418008 JX418011 A. diluta subsp. chrysantha JQ404519 JQ404668 JQ404900 A. diluta subsp. diluta JQ404519 JQ404668 JQ404900 A. graminea JQ404527 AY120360 JQ404781 A. hookeri subsp. cummingiana JQ404528 JQ404674 JQ404782 JQ404904 A. inodora JQ404529 JQ404697 JQ404810 JQ404931 JQ405047 A. isabellana JQ404531 JQ404675 JQ404783 JQ404905 JQ405018 A. ligtu subsp. simsii JQ404536 JQ404679 JQ404788 JQ404909 JQ405022 A. magnifca var. magnifca JQ404540 JQ404682 JQ404791 JQ404912 JQ405025 A. orchidioides JQ404686 JQ404794 JQ404915 JQ405030 A. pallida JQ404547 JQ404687 JQ404795 JQ404916 JQ405031 A. patagónica JQ404548 AY120362 JQ404796 JQ404917 JQ405032 A. pelegrina JQ404549 AY120363 JQ404797 JQ404918 A. philippii JQ404552 JQ404689 JQ404799 JQ404920 JQ405034 A. presliana subsp. presliana JQ404555 JQ404690 JQ404800 JQ404921 JQ405036 A. pseudoespathulata JQ404556 JQ404691 JQ404801 JQ404922 JQ405037 A. psittacina JQ404557 AY120364 JQ404802 JQ404923 JQ405039 A. pygmaea JQ404559 AY120365 JQ404804 JQ404925 JQ405041 A. versicolor JQ404580 JQ404707 JQ404819 JQ404941 Bomarea B. boliviensis JQ404589 AY120368 JQ404827 JQ404947 B. involucrosa JQ404605 JQ404727 JQ404841 JQ404960 EU159945 B. macrocephala JQ404607 AY120367 JQ404842 JQ404961 B. ovallei JQ404614 AY120369 JQ404849 JQ404965 EU159950 B. ovata JQ404615 JQ404733 JQ404850 JQ404966 B. patinii subsp. patinii JQ404619 JQ404737 JQ404854 JQ404970 EU159951 B. salsilla JQ404625 JQ404740 JQ404857 JQ404973 B. setacea JQ404627 EU159967 EU159953 B. stans JQ404632 JQ404362 Drymophila D. cyanocarpa JQ404644 JN417503 JQ404872 JQ404983 D. moorei JQ404645 JN417504 JQ404875 JQ404985 Luzuriaga L. marginata JQ404651 JQ404758 JQ404878 JQ404988 JQ405066 L. parvifora JQ404655 JQ404760 JQ404883 JQ404993 L. radicans JQ404657 JQ404763 JQ404886 JQ404996 JQ405067 Smilax S. aspera HM850359 JF461378 KJ719935 ecological niches. Nevertheless, the South American taxa Chromosome number cover a higher latitudinal and longitudinal extension in comparison with taxa from Oceania which present reduced Three diferent chromosome numbers are recorded within geographical extension. the Alstroemeriaceae family (Table 1). Alstroemeria has a

1 3 Cytogenetics of Alstroemeriaceae Page 9 of 22 34

Table 3 Location of chromosome satellites in Alstroemeriaceae species. m metacentric; st subtelocentric; t telocentric. p short arm; q long arm; cs secondary constriction (based on references in Table 1) Chromosome pair and satellite location Species 1 2 3 4 5 6 7 8 9 10

Alstroemeria andina var. venustula t(p) t-st(p) t(p) A. angustifolia subsp. angustifolia t(p) t(p) t(p) t(p) A. aurea t(p) t(p) t(p) t(p) A. diluta subsp. chrysantha t(p) st(p) A. graminea st(p) t(p) st(p) A. hookeri subsp. hookeri (coast) t(p) st(p) A. hookeri subsp. hookeri (valley) st(p) st(p) st(p) A. hookeri subsp. recumbens st(p) A. hookeri subsp. maculata t(p) st(p) m(p) A. hookeri subsp. cummingiana st(p) A. inodora t(p) t(p) t(p) t(p) A. ligtu subsp. ligtu m(p) m(p) t(p) t(p) sm(p) t(p) A. ligtu subsp. ligtu t(p) t(p) st(p) A. ligtu subsp. simsii m(p) t(p) t(p) t(p) A. isabellana sc-sm(q) t(p) t-st(p) t-st(p) t(p) A. magnifca subsp. magnifca st(p) st(p) st(p) A. magnifca var. magenta t(p) A. magnifca var. tofoensis t(p) t(p) A. marticorenae t(p) st(p) A. presliana subsp. presliana sm(p) st(p) A. presliana subsp. australis m(p) A. patagonica st(p) sm(p) A. versicolor st(p) st(p) A. pelegrina st(p) st(p) st(p) sm(p) A. philippii t(p) t(p) A. pygmaea t(p) t(p) t(p) A. psittacina sc-st(q) t(p) t(p) t(p) A. psittacina t(p) t(p) t(p) t(p) Bomarea ovallei t(p) B. ovallei st(p) B. salsilla t(p) st(p) st(p) B. patinii subsp. patinii t(p) sm(p) L. parvifora sm(p) number 2n = 16, Bomarea 2n = 18, and Luzuriaga 2n = 20. of the family, although with low frequency. Thus, one or In the case of Drymophila, numbers 2n = 20 have been two Bs were observed in one accession of A. andina var. described for D. moorei and 2n = 24 for D. cyanocarpa. venustula, and three Bs were described in one accession of However, the number 2n = 20 has been the most accepted for A. angustifolia subsp. angustifolia. the Drymophila genus. All species are diploids, except for Bomarea hirtella, which has been reported as a tetraploid, Karyotype morphology with 2n = 36. The series of basic chromosome numbers accepted so far within the family is x = 8, 9, and 10. Geo- The karyotype structure of Alstroemeriaceae species shows graphically, the variation in 2n numbers of South American diferent levels of variation according to their genus, and taxa (2n = 16, 18, and 20) is higher than Oceania (solely the diferences can be based on chromosome morphology, 2n = 20). Supernumerary chromosomes or B-chromosomes relative length of the chromosomes within the karyotype have also been observed within the South American taxa or total haploid length (THL) (Table 1). In Bomarea, the

1 3 34 Page 10 of 22 P. Jara-Seguel et al.

Table 4 Meiotic chromosomes of Alstroemeriaceae species. I univalent; II bivalent, AI anaphase I; MI metaphase I; TI telophase I Species n Chromosome confguration Chiasmata frequency References

Alstroemeria andina var. venustula 8 8 II Typical chiasmata were almost undetect- Sanso and Wulf (2007) able. Bridges and fragments in AI-AII A. aurea 8 8 II Almost two chiasmata in diakinesis or Hunziker and Xifreda (1990) metaphase A. hookeri subsp. cummingiana 8 1 or more II in MI Bridges and fragments in TI Sanso (2002) A. isabellana 8 8 II Almost two chiasmata in diakinesis or Sanso and Hunziker (1998) metaphase A. pallida 8 7 II + 2 I Sanso (2002) A. patagonica 8 8 II Two or three chiasmata Sanso (2002) A. pseudospathulata 8 8 II Two or three chiasmata Sanso (2002) A. psittacina 8 8 II Almost two chiasmata in diakinesis or Sanso and Hunziker (1998) metaphase A. psittacina 8 8 II Almost two chiasmata in diakinesis or Hunziker and Xifreda (1990) metaphase 9 8 II + 2 I Sanso and Hunziker (1998) B. edulis 9 9 II Hunziker and Xifreda (1990) B. boliviensis 9 8 II + 2 I Sanso and Hunziker (1998) B. setacea 9 9 II Hunziker and Xifreda (1990)

Table 5 C-band distribution Genus/Species 2n Heterochromatin distribution RLn (%) in karyotypes of the (C-bands) (C-bands) Alstroemeriaceae species studied to date. T number of T I P TB telomeric bands; I number of interstitial bands; P number Alstroemeria angustifolia subsp. angustifolia 16 6 13 0 19 12.8 of proximal bands; TB total A. aurea 16 9 8 0 17 16.8 RLn number of C-bands; A. graminea 16 3 2 2 7 4.5 (%) relative length of the A. inodora C-bands. Data were taken 16 3 13 7 23 1.5 from Buitendijk and Ramanna A. ligtu subsp. incarnata 16 10 21 1 32 22.1 (1996), Buitendijk et al. (1998), A. ligtu subsp. ligtu 16 9 21 2 32 12.9 Jara-Seguel et al. (2004, 2005) A. ligtu subsp. simsii 16 11 22 5 38 19.1 A. magnifca subsp. magnifca 16 4 6 0 10 6.5 A. pelegrina 16 5 1 0 6 3.3 A. philippii 16 2 0 0 2 2.0 A. psittacina 16 1 10 0 11 1.0 Bomarea ovallei (≡ Leontochir ovallei) 18 4 4 1 9 20.0 karyotypes did not vary signifcantly in morphology among taxa of Alstroemeria (belonging to 18 species) have from species. The six species studied so far have from 22.2 to 37.5 to 75% of metacentric and/or submetacentric chromo- 55.5% of metacentric and submetacentric chromosomes somes within the karyotype, whereas the remaining percent- within the karyotype, whereas the remaining percentage is age is represented by subtelocentric and/or telocentric chro- represented by subtelocentric and telocentric chromosomes mosomes. The extremes were A. graminea with karyotype depending on the species. The extremes were B. boliviensis formula n = 1m + 2sm + 4st + 1t = 8, and A. ligtu subsp. ligtu (karyotype formula: n = 2m + 5st + 2st − t = 9) and B. invo- with karyotype formula n = 4m + 1sm + 3t = 8. In the case lucrosa (karyotype formula: n = 2m + 3sm + 4st = 9), respec- of Luzuriaga, the karyotypes have a large pair 1 (relative tively. The genus Alstroemeria, with described karyotype for length 22–23%), almost doubling the smallest pairs in size. 27 taxa, showed higher karyotype variation at an interspe- All three species studied for Luzuriaga have from 70 to 90% cifc level in comparison with Bomarea. The karyotypes of of metacentric and/or submetacentric chromosomes within Alstroemeria have a large pair 1 (relative length between 27 the karyotype, whereas the remaining percentage is repre- and 30%), almost doubling the smallest pairs in size. All 27 sented by subtelocentric chromosomes. The extremes were

1 3 Cytogenetics of Alstroemeriaceae Page 11 of 22 34

Fig. 1 Bayesian inference phylogenetic based on combined are found above the branches. Haploid chromosome number (n), most analysis (rDNA and cpDNA) for taxa of the Alstroemeriaceae fam- frequent haploid karyotype formula (HKF), and mean haploid set ily. The subfamilies are indicated by vertical lines and Smilax aspera length (THL in µm) of each genus of Alstroemeriaceae were superim- (Smilacaceae) was used as the outgroup. Posterior probability values posed onto a phylogenetic framework

Fig. 2 Map showing geographical distribution of the four genera belonging to Alstroemeriaceae. Yellow circle, Alstroemeria; blue circle, Bomarea; green circle, Drymophila; and fuchsia circle, Luzuriaga 1 3 34 Page 12 of 22 P. Jara-Seguel et al.

L. parvifora with karyotype formula for one metaphase 41.5 and 70 pg). Unfortunately, nuclear DNA content is a n = 4m + 3sm + 3st = 10, and L. radicans with karyotype for- feature that has still not been studied for a large part of the mula n = 3m + 6sm + 1st = 10. In the case of Drymophila spe- Alstroemeria species, nor for species of Bomarea, Luzuriaga cies the karyotype morphology has not yet been described. and Drymophila. The correlation between THL and DNA Another feature that has been studied for species of the C-values was signifcant and positive (r = 0.92; p < 0.05) for three genera of Alstroemeriaceae is the total haploid set the seven Alstroemeria species where DNA C-values and length (THL), which displays high variability. As a refer- karyotypes have been studied. Conversely, the correlation ence, we estimated a mean THL = 76.31 ± 18.27 µm that is was signifcant and negative among C-values and MCA representative of the family, with a range of THL that varies (r = 0.72; p < 0.05) as well as between C-values and CVCL between 42.5 µm in B. salsilla and 112.0 µm in A. pygmaea. (r = 0.95, p < 0.05) for the same species. In a more detailed analysis, 17 taxa (15 taxa of Alstroemeria and two of Bomarea) have THL values higher than the mean Karyotype asymmetry estimated here for the family. In the case of the genus Luzu- riaga represented by L. radicans, its THL = 48.7 µm was According to our analysis based on the calculation of the lower than the mean value of the family. MCA (mean centromeric asymmetry) and CVCL (coef- cient of variation of chromosome size) (Fig. 3, Table 1), Nuclear DNA content in general the family exhibited wide variation in karyotype asymmetry, and more specifcally in the coefcient of varia- Nuclear DNA content (or C-value) is other characteristic tion of the chromosome length (CVCL). The two out-groups studied for 13 Alstroemeria species distributed in Chile having symmetric [Krameria cistoidea (Krameriaceae), and Brazil (Table 1). The higher 2C-value was described haploid karyotype formula n = 6m] and asymmetric [Mier- in Alstroemeria ligtu subsp. incarnata with 2C = 70 pg, sia chilensis () haploid karyotype formula: and the lower 2C-value was described in A. philippi with n = 1m + 9t = 10] karyotypes denote the characteristic 2C = 41.5 pg, both in Chile. Brazilian species show low asymmetry of the Alstroemeriaceae species. In this plot, variation in 2C-values (range between 49.8 and 55.5 pg), groups of species can be identifed. Thus, the dots repre- whereas Chilean species show high variation (range between senting Bomarea species are grouped together, showing

Fig. 3 Analysis of karyotype asymmetry in Alstroemer- iaceae according to Peruzzi and Eroglu (2013). The non-related outgroups are represented by: Kci Krameria cistoidea (Krameriaceae) (2n = 12) with symmetric karyotype (haploid karyotype formula: n = 6 m; Palma-Rojas et al. 2019); and Mch Miersia chilensis (Alli- aceae) (2n = 20) with asymmet- ric karyotype (haploid karyo- type formula: n = 1 m + 9t = 10; Escobar et al. 2012). Yellow circle, Alstroemeria; blue circle, Bomarea; and fuchsia circle, Luzuriaga

1 3 Cytogenetics of Alstroemeriaceae Page 13 of 22 34 low variation in both MCA (between 51 and 57) and CVCL Interestingly, in some species SATs can appear in the short (between 14 and 27) values and displaying less asymmet- arm of metacentric pair 1 or 2. ric karyotypes than Alstroemeria and Luzuriaga. The plot refects the similarities in both chromosome morphology and Meiotic chromosomes relative length observed among Bomarea species, with all of them presenting unimodal karyotypes. Meiosis has been studied in 11 Alstroemeriaceae species, The three Luzuriaga species for which the karyotypes are seven Alstroemeria species (including two subspecies) and known display low variation in MCA (between 31 and 40) three Bomarea species. Some details on meiosis published in and CVCL (between 48 and 54) values. The dots represent- the literature are summarised in Table 4. The studies report- ing the asymmetry values of Luzuriaga are also in a cluster. ing the n chromosome number in meiosis have been useful to Interestingly, the dots of the Luzuriaga and Alstroemeria corroborate the 2n chromosome numbers studied in species species are grouped together (Fig. 3), refecting afnity in of both genera. chromosome morphology and relative length, both genera In general, the meiosis is regular, showing mostly biva- having bimodal karyotypes. On the other hand, Alstroemeria lents in all species studied, with univalent formations species have highly asymmetric karyotypes, with a moderate described only infrequently. An exception is A. hookeri variation in MCA (between 27 and 55) and high variation in subsp. cummingiana in which meiotic irregularity stages CVCL (between 19 and 64). With regard to CVCL values, were observed. In addition, two chiasmata have been groups of species can be identifed; thus group 1 has high described as the most frequent confguration in meiosis, CVCL values, group 2 has intermediate CVCL values, and and few cases with three chiasmata formations have been group 3 has low CVCL values. reported. In two species (e. g., A. hookeri var. cummingiana Location of chromosome satellites and A. andina subsp. venustula) bridges, fragments and/or lagging chromosomes have been observed in metaphase I or in anaphases I and II. Moreover, micronuclei in the state Location of chromosome satellites (SAT) is another karyo- of dyads or tetrads have also been observed in metaphase I type feature studied in Alstroemeriaceae. Chromosome pairs and anaphases I and II. of Alstroemeriaceae species carrying SATs are shown in Table 3, which was constructed based on references shown in Table 1. In Bomarea, SATs have only been observed in C‑banding and FISH banding patterns three species, B. ovallei, B. salsilla, and B. patinii subsp. patinii. In the case of one population of B. ovallei var. oval- The distribution of constitutive heterochromatin in Alstro- lei that inhabits Carrizal Bajo (28°08´S; 71°07´W), northern emeriaceae has been studied using the C-banding method Chile (28°S), SATs were observed in the short arm of telo- for nine Alstroemeria species from Chile and Brazil (nine centric pair 2, whereas in other geographically near popula- species and fve subspecies) and one species of Bomarea (B. tions of B. ovallei located in Llanos de Challe National Park ovallei) (Table 5). Alstroemeria presents a wide variation in (28°07´S; 71°06´W), SATs were observed in subtelocentric chromosome banding, especially in terms of location, size pair 4. In the case of B. salsilla, two populations have been and relative length of the C-bands. The higher relative length studied, but SATs have only been observed in one of them, value of the C-bands has been described in A. ligtu subsp. in the short arms of chromosome pairs 2 (telocentric), 3 incarnata from Central Chile (22.1% of relative length of and 7 (both subtelocentric). In B. patinii subsp. patinii from the C-bands), whereas the lower value was found in A. psit- Colombia (4°N) SATs were present in telocentric pair 1, tacina from Brazil (1.0% of relative length of the C-bands). which is equivalent in shape and close in size to pair 2 of The high values of relative length of the C-bands can be B. ovallei. related to the presence of large blocks or small dispersive but Within the genus Luzuriaga, satellites have been observed numerous blocks of constitutive heterochromatin in some in L. radicans and L. parvifora. In the case of L. radicans, chromosome arms, whereas low values of relative length one satellite was observed in the short arm of submetacentric of the C-bands are related to small blocks located in some pair 6, whereas in L. parvifora the satellite was observed in chromosome arms. A. ligtu subsp. simsii is the subspecies the short arm of submetacentric pair 10. with the highest polymorphism in the C-banding pattern (up The majority of studies describing the location of SATs to 38 bands can be counted within the karyotype) displaying location have focused on Alstroemeria. To date, 14 studies telomeric, interstitial and proximal bands. In the case of the including 18 species, of which seven include subspecies, Bomarea genus, the only studied species was B. ovallei from have been published (Table 3). In general, the most frequent northern Chile, which had a high relative C-band length of satellite-bearing chromosomes at interspecifc levels are 20%. This species showed large blocks of constitutive het- pairs 3, 4, 5 and/or 6, which are telocentric or subtelocentric. erochromatin in several chromosomes within the karyotype.

1 3 34 Page 14 of 22 P. Jara-Seguel et al.

Studies using molecular cytogenetic methods in Alstro- and Rodriguez 2006; Zuloaga et al. 2008; Alzate 2016). In emeriaceae are scarce in wild species. Table 6 summa- general, Alstroemeriaceae, with their four genera included in rises some data published in the literature. Currently, data two subfamilies, are characterised cytogenetically by attrib- on physical mapping by fuorescent in situ hybridisation utes such as: (i) the presence of three chromosome num- (FISH) of ribosomal genes (18-25S rDNA, 45S rDNA and bers (16, 18 and 20); (ii) variation or stability in karyotype 5S rDNA) and repetitive DNAs (A001—1DNA) have been characteristics such as morphology, asymmetry, location of performed for ten Alstroemeria species, in addition to the chromosome satellites and total haploid length depending on BAC-FISH and genomic in situ hybridisation (GISH) results the genus; (iii) stability in meiotic behaviour but with two published so far. species showing irregularities; (iv) polymorphism in C and The analysis of the physical location of the ribosomal FISH banding as performed in some species of Alstroemeria genes in Alstroemeria shows that these cytogenetic mark- and Leontochir (≡ Bomarea ovallei), and (v) interspecifc ers present a high polymorphism, with ribosomal clusters variation in DNA C-values as was evidenced in some Alstro- 18-25S rDNA and 5S rDNA being the most used. These emeria species. These cytogenetic attributes are discussed markers can vary between 5 and 17 sites of 18-25S rDNA here, including a dataset gathered for 50 wild species and 43 per genome, whereas the 5S rDNA vary between 4 and 18 reports published for the family. sites per genome, with the terminal location as the most frequent within the karyotype for these ribosomal markers Chromosome number (Table 6). The Bayesian inference phylogenetic tree shown in Fig. 1 supports the subfamilial and generic divisions within Alstro- Discussion emeriaceae previously described by other authors, some of them including a hypothesis on monophyly for the family The cytogenetic diversity described within Alstroemeriaceae (Aagesen and Sanso 2003; Chacón et al. 2012b). This phy- is concentrated mainly in South America where a higher logenetic framework is also consistent with some distinctive number of genera and species also occur in a latitudinal cytogenetic features of each genus, among them the base range covering ca. 6000 km from 2°N to 51°S along the chromosome number. A recent phylogenetic reconstruction continent (Fig. 2 and Table 1). This observation is concur- of the ancestral chromosome number for Alstroemeriaceae rent with the more complete phylogenetic reconstruction was performed and included within a robust analysis car- published so far for the family based on DNA sequences ried out for 318 angiosperm families (Carta et al. 2020). In (Chacón et al. 2012b), as well as with various floristic that analysis, the numbers n = 10, 9 and 7 were suggested as reports analysing species diversity (Sanso 1998; Conran ancestral for the family, having been inferred from the most and Cliford 1998; Arroyo-Leuenberger 2001; Hofreiter recent common ancestors under Bayesian optimisation and

Table 6 Molecular cytogenetic markers used in Alstroemeria species I interstitial; P pericentromeric; S satellite; SC secondary constric- and genomic distribution of the positive bands. Chromosome loca- tion. Data were taken from Kamstra et al. (1999, 2004), Zhou et al. tions of the bands are abbreviated as follows: T terminal/subterminal; (2003), Baeza et al. (2007a), Chacón et al. (2012a)

Species 18-25S rDNA 45S rDNA 5S rDNA A001-1 DNA Other markers No. of sites Location No. of sites Location No. of sites Location No. of sites Location

Alstroemeria aurea 16–17 T, I, S 6–7 T, I, P 9 T, I BAC-ISH/GISH/ Chrom. paint- ing A. hookeri 7 T, SC 18 T, I 4 T, I A. inodora BAC-ISH/GISH/ Chrom. paint- ing A. ligtu 9 T, I, S 13 T, I, P, SC 0 Chromosome painting A. longistaminea Mart 6 T ND A. pelegrina 5 T, S 4 I, P 0 A. presliana 12 T, I, P, S 11 T, I, P 0 A. pulchella 10 T, I 5 T, I ND A. af. rupestris 9 T, I 8 T, I ND A. stenopetala Seub 10 T ND 1 3 Cytogenetics of Alstroemeriaceae Page 15 of 22 34 exhibiting respective posterior probability values of 0.228, evident that Alstroemeriaceae are among the families with 0.218 and 0.185. It is noteworthy that the numbers 10 and 9 lower diversity in chromosome number in comparison with are present in three of the four genera of the family. other families of the order, despite their wide intercontinen- The chromosome number is conservative within the tal distribution. Possibly, the species diversifcation within Luzuriagoideae subfamily, including Luzuriaga and Dry- the family has occurred mainly due to changes in chromo- mophila (both 2n = 20), whereas within Alstroemerioideae some morphology and size (absolute or relative length) as it is variable, with numbers 2n = 18 in Bomarea and 2n = 16 described in Fig. 3, and secondarily through chromosome in Alstroemeria. For this reason, the 2n number is a cytoge- number modifcations according to the evidence provided netic feature that has been taxonomically used to separate here. Alstroemeria from Bomarea (Hunziker and Xifreda 1990; Sanso and Hunziker 1998; Sanso 2002; Jara-Seguel et al. Karyotype morphology 2004; Palma-Rojas et al. 2007) and is also consistent with taxonomic treatments among both genera based on morphol- Studies on karyotype morphology in Alstroemeriaceae have ogy (Sanso 1998). Additionally, the chromosome number included single species or groups of them. The purpose of added to morphological characteristics has facilitated the these reports has been to analyse the karyotype structure taxonomic separation of Alstroemeria and Bomarea from focused on understanding cyto-evolutionary and taxonomic Luzuriaga and Drymophila (Manadue and Orchard 1985; aspects. In general, constancy in chromosome number and Löve 1985; Wiltshire and Jackson 2003; Jara-Seguel et al. uniformity or conservatism of karyotype morphology within 2010). The three chromosome numbers of the family have each genus has been reported by some authors (Buitendijk a wide distribution in various environments along the geo- and Ramanna 1996; Sanso and Hunziker 1998; Sanso 2002; graphical range (e.g., arid, humid and cold environments) Palma-Rojas et al. 2007; Jara-Seguel et al. 2010). In our and only one of them occurs also in forests in Oceania (Aus- work, and based on a wide dataset, it was possible to visual- tralia, Tasmania, and New Zealand) (Fig. 2). Bomarea is ise a comparatively high karyotype diversity within the fam- distributed from South America to Central America, but the ily when analysing various cytogenetic characteristics such available cytogenetic data have a northern limit in Colombia, as karyotype morphology and asymmetry, satellite loca- with three species studied that inhabit there (Table 1, Fig. 2). tion (Table 1) and cytomolecular markers (Tables 5 and 6). Currently, cytogenetic data are not available for Bomarea Alstroemeria was the cytogenetically more variable genus. species from Central America (around three species, Alzate Of all the parameters used to describe karyotypes, asym- et al. 2008); therefore this group of tropical species should metry indices provide a more objective and simultaneous be studied cytogenetically to increase the geographical cov- comparison among species, representing a quantitative erage up to the northern limit, including chromosome num- analysis that co-relates two parameters (MCA and CVCL) ber and other karyotype features. as proposed by Peruzzi and Eroglu (2013). Based on our A query arises as to whether Alstroemeriaceae have high asymmetry analyses, it is possible to detect high interge- or low diversity in chromosome number compared to other neric karyotype variation mainly between Alstroemeria and families within the Liliales order, which could also extend to Bomarea, as well as among some species within each genus, its wide geographical distribution along two continents. Pre- but most clearly observed within Alstroemeria. According vious reports pointed out that the family exhibits little vari- to Fig. 3, four clearly separated groups on the plot were ation in chromosome number compared to its sister family observed, three encompassing Alstroemeria and Luzuriaga Colchicaceae, which has almost 30 diferent 2n chromosome species and a fourth made up of Bomarea species. Based on numbers (Chacón et al. 2012b, 2014). We included other these analyses, we are able to support previous assumptions families in this comparison, i.e. Liliaceae, with 20 diferent regarding variations in gross karyotype morphology within chromosome numbers (Peruzzi et al. 2009), Melanthiaceae Alstroemeria such as has been described by some authors with 10 diferent chromosome numbers (Pellicer et al. 2014), (e.g., Buitendijk and Ramanna 1996; Sanso and Hunziker and Smilacaeae with seven diferent chromosome numbers 1998) and also to confirm the karyotype conservatism (Vijayavalli and Mathew 1989). These four families show a described for Bomarea (Hunziker and Xifreda 1990; Sanso high diversity in chromosome number, including diploids and Hunziker 1998; Palma-Rojas et al. 2007; Baeza et al. and polyploids, and have intercontinental distribution. In 2008a) and Luzuriaga (Jara-Seguel et al. 2010). A major contrast, other families within Liliales with restricted geo- part of the Alstroemeria species showed wide diversity in graphical distribution showed lower diversity in chromo- karyotype asymmetry due to the high variation observed in some number, such as Philesiaceae and Rhipogonaceae, CVCL values (range between 19 and 64) accompanied by a both with only one chromosome number (Beuzenberg and lower variation in MCA values (range between 27 and 55). Hair 1963); Jara-Seguel and Fuentes-Ramírez 2007; Daw- Thus, the species are ordered in groups where the opposite son 2008). Thus, according to these cytogenetic data, it is extremes are A. graminea (MCA = 53, CVCL = 64) from

1 3 34 Page 16 of 22 P. Jara-Seguel et al. northern Chile, which exhibits higher karyotype asymmetry, among Alstroemeria species, thus supporting the informa- and three subspecies of A. ligtu (MCA = 39; CVCL = 19 and tion obtained through quantitative cytogenetic methods. As 21) from southern Chile with lower karyotype asymmetry. additional evidence related to the meiotic studies on gam- These aspects were previously described for the two spe- ete formation summarised in Table 4, regularity in bivalent cies on the basis of karyotype morphology and C-banding or univalent formation and chiasmata frequency in almost patterns (Buitendijk and Ramanna 1996; Jara-Seguel et al. eight Alstroemeria species studied so far has been observed. 2004). In addition, analyses on karyotype asymmetry have Thus, the chromosome stability during gamete formation is also been used to discriminate among subspecies of A. pres- maintained despite the apparent occurrence of chromosome liana, A. ligtu and A. magnifca, respectively, showing dif- rearrangements as discussed by Stebbins (1971) in other ferences among the taxa (Baeza al. 2015a, b). In the case of groups. However, some meiotic irregularities observed subspecies of A. magnifca, and A. presliana, some taxo- in A. hookeri var. cummingiana and Alstroemeria andina nomic changes have been suggested to be based on karyo- var. venustula could be an additional source of evidence type asymmetry and morphology (Baeza et al. 2015a, 2018a; to support that chromosome modifcations are in process Finot et al. 2018). However, phylogenetic studies based on (e.g., inversions, insertions, translocations) thus afecting DNA sequences are required to prove these cyto-taxonomic the meiosis during pollen formation (Sanso 2002; Sanso hypotheses more robustly (Fryday et al. 2020; Jara-Arancio and Wulf 2007). These irregularities could also generate et al. 2019; Rehner and Samuels 1994). In our analyses we diferent levels of sterility when a heterozygote condition also observed that Brazilian species are grouped with some occurs as a result of chromosome rearrangements (Gallardo Argentinian and Chilean species, all presenting similarities 2011). Chromosome heterozygocity has been described in in karyotype asymmetry. This grouping is consistent with a mitotic homologous pairs in some Alstroemeria species ana- phylogenetic hypothesis suggesting an evolutionary relation- lysed by karyotypes, C-banding and FISH (Buitendijk and ship among ancestral (Chilean and Argentinian species) and Ramanna 1996; Baeza et al. 2007a, 2018b), suggesting that derivative (Brazilian species) clades within Alstroemeria the occurrence of meiotic alterations could be possible in (Chacón et al. 2012a), and is also supported by the presence wild species. of a conservative karyotype asymmetry among both lineages The uniformity in karyotype asymmetry described in the as shown in this study. three Luzuriaga species studied to date revealed that fne The high variation revealed in our analyses on karyotype chromosome changes have occurred during their evolution- asymmetry suggests that chromosome modifcations exist in ary history but have not modifed the karyotype morphology Alstroemeria, evidencing their previously described genome too much (Jara-Seguel et al. 2010). Interestingly, similari- plasticity (Buitendijk and Ramanna 1996; Buitendijk et al. ties exist in terms of karyotype asymmetry among Luzu- 1997). Changes could even still be occurring in the popula- riaga and some Alstroemeria species, thus forming a group tions, as has been suggested in studies of gross chromosome despite diferences in their chromosome number. As an addi- morphology in some species (Sanso 2002; Cajas et al. 2009; tional observation, the presence of bimodal karyotypes is a Baeza et al. 2015a, b). In this regard, the current cytogenetic common and remarkable feature in both Alstroemeria and evidence suggests the occurrence of chromosome rearrange- Luzuriaga, while Bomarea has unimodal karyotypes (Sanso ments in some karyotypes, which has been revealed through 2002; Palma-Rojas et al. 2007; Jara-Seguel et al. 2010). quantitative analyses using Karyo-Idiograms (Jara-Seguel Karyotype morphology of Alstroemeriaceae—specifcally 2001) and asymmetry analysis (Cajas et al. 2009), highlight- Alstroemeria and Luzuriaga—shows a similar structural ing pericentric inversions as the main changes. Addition- pattern compared with other Liliales families. For example, ally, the use of C-banding and FISH methods has revealed in Smilacaceae the karyotypes of some diploid species of that pericentric inversions would be associated to insertions, the genus Smilax are strikingly asymmetrical, with a largest translocations, and fusions of DNA sequences (Tables 5 and pair 1 almost doubling the size of the smallest (Vijayavalli 6; Buitendijk and Ramanna 1996; Buitendijk et al. 1998; and Mathew 1989), similar to what was observed in Alstro- Kamstra et al. 1999, 2004; Zhou et al. 2003; Jara-Seguel emeria and Luzuriaga. However, in the above-mentioned et al. 2004; Baeza et al. 2007a). In a recent study, phyloge- study we evidenced some diferences with regard to those netic signals have suggested the direction of the chromosome described for Alstroemeria and Luzuriaga. These were changes among the lineages, ofering a stronger hypothesis inferred by observation of karyotype formulas, where the regarding genome evolution in the case of Alstroemeria greater part of the Smilax species present between 40 and (Chacón et al. 2012b). In addition, studies based on DNA 50% of metacentric and submetacentric chromosomes within fngerprinting (AFLP and RAPD; Han et al. 2000; Baeza the karyotype, and the remaining chromosomes are subte- et al. 2007b; Finot et al. 2018) and cytomolecular meth- locentric or telocentric. On the other hand, Lilaceae with a ods (genomic slot blot, southern blot, and GISH; Kuipers large cytogenetic dataset display variable karyotype asym- et al. 1997) reveal the existence of genome polymorphism metry among genera (Peruzzi et al. 2009) and almost fve of

1 3 Cytogenetics of Alstroemeriaceae Page 17 of 22 34 them show several similarities in MCA and CVCL values in for plants (Singh 2003; Heslop-Harrison and Schwarzacher comparison with Alstroemeriaceae. In general, several com- 2011). In certain Alstroemeria species fuorescent meth- mon patterns of karyotype asymmetry are evident among ods have revealed that some satellites are co-localised with some species of both families despite their taxonomic sepa- rDNA sites 45S (18S-25S) or 5S (Table 5). The rDNA sites ration, possibly due to a basic common genomic architecture exhibit polymorphism at an intra- and inter-specifc level in within the Liliales order. the Alstroemeria species studied. In addition, the presence of The Bomarea species studied here evidence general telomeric sites with interstitial location in chromosome arms uniformity in karyotype morphology and low asymmetry, suggests that chromosome fusions would have occurred which is supported by the respective values of MCA (range during their evolution, as was revealed in A. cf. rupestris between 51 and 58) and CVCL (range between 14 and 27). (Chacón et al. 2012a). These observations suggest a high These are very close among species, forming a cohesive dynamic of the ribosomal compartment in which fne chro- group that includes B. ovallei species previously classifed as mosome rearrangements would have been crucial to generate a separate monotypic genus named Leontochir. Such karyo- changes in the location of chromosome satellites as well as type uniformity is concurrent with regularities in the meiosis in the respective secondary constrictions containing rDNA. of some Bomarea species, where bivalent or univalent for- In Bomarea the observation of satellites has proven dif- mation was observed in the three species studied (Table 3). fcult for populations of some species (B. edulis, B. seta- However, the karyotype uniformity described here for six cea, B. salsilla, and B. involucrosa) (Sanso and Hunziker Bomarea species is inconsistent with topologies obtained 1988; Palma-Rojas et al. 2007) whereas other populations in phylogenetic reconstructions previously reported for the do carry satellites, as has been described in B. salsilla genus, which includes three species studied in our analy- (Baeza and Silva 2016). The use of protocols including ses but which are contained in three separate clades (Alzate diferent antimitotic reagents (8-Hydroxyquinoline and et al. 2008). Such inconsistence is feasible in that chromo- Colchicine) may explain such diferences afecting chro- some morphology and nucleotide sequences of the nuclei matin packing in the chromosomes. In addition, discrep- or plastid genomes represent diferent organisational levels ancy exists among some authors regarding the location and are also evolutionarily independent. Nevertheless, more of chromosome satellites within the karyotypes, which Bomarea species should be studied cytogenetically to supply could simply be an indication of polymorphism among additional information that would make it possible to under- populations of each species evidencing the plasticity of stand their chromosome evolution along their wide distribu- their satellite compartment along their geographical dis- tional range and address the possibility of superimposing it tribution (Palma-Rojas et al. 2007; Baeza et al. 2008a, onto the phylogenetic framework. 2012; Baeza and Silva 2016). In general, the short arm of telocentric pair 2 is the more frequent satellite carrier, Satellite location and rDNA being present in two species (B. salsilla and B. ovallei), while other chromosomes carrying satellites are pairs 1, The presence of satellites is another cytogenetic feature that 3, 4 or 7 depending on the species. In addition, it is pos- has been studied when describing karyotype morphology in sible to observe that the location of chromosome satellites Alstroemeriaceae (Table 3). The higher variation in chro- in pair 3 of B. ovallei and pair 4 of B. salsilla are shared mosome location and number of satellites was found in the with most of the Alstroemeria species, evidencing a con- genus Alstroemeria with 18 species studied (including nine servative location of satellites as might be expected among subspecies and two varieties), with the short arms of telo- sister genera with common ancestors (Table 3). centric pairs 3, 4, 5, and/or 6 being the most frequent carriers In the Luzuriaga species, only one chromosome carrying depending on the species. This conservative chromosome a satellite has been described (Table 3). So in L. radicans location of satellites in some pairs within the karyotypes of submetacentric pair 6 carries a satellite in its short arm, Alstroemeria could be a characteristic retained from their whereas in L. parvifora the satellite is located in the short ancestors (Jara-Seguel 2001). Exceptions to this pattern were arm of submetacentric pair 10. However, it is possible to A. hookeri subsp. cummingiana, A. presliana subsp. presli- suppose that these submetacentric chromosomes with satel- ana and A. presliana subsp. australis with satellites located lites present in the current species of Luzuriaga may have in other chromosome pairs (including some metacentric) been inherited from an ancestor and that through rearrange- difering from those mentioned above. The evolutionary ment their relative length has changed, but not their shape dynamic of chromosome location and number of satellites (or centromere position). Such submetacentric chromosomes in Alstroemeria could be linked to the evolution of rDNA carrying satellites are located in diferent positions within in that secondary constrictions located in sub-satellite chro- the respective karyotypes. So during the evolutionary pro- mosome segments could be sites containing such sequences cess the satellite and secondary constriction would have according to patterns of chromosome organisation described

1 3 34 Page 18 of 22 P. Jara-Seguel et al. conserved their subtelomeric location in the short arm of specifc DNA sequences, AT/GC ratio) for species of the dif- the chromosome (Jara-Seguel et al. 2010). ferent environments of mainly Brazilian and Chilean species Fluorescence methods have proved useful to describe the (Kuipers et al. 1997; Buitendijk et al. 1997). However, some structure and dynamics of the karyotypes in some Alstro- studies in plants suggest that soil properties such as nitro- emeria species and for this reason should be used in more gen (N) and phosphorous (P) content are essential nutrients taxa of the family. This could facilitate the understanding of for the production of nucleotides and proteins, but these are the cyto-evolutionary process of the ribosomal DNA as well subject to ecological limitation in most ecosystems (Šmarda as the other DNA fractions in the four genera of the family et al. 2013; Kang et al. 2015). Thus, it might be useful for (Baeza et al. 2007a; Chacón el at. 2012b; Finot et al. 2018). research on genome structure and dynamics described for Alstroemeriaceae species to focus on determining mecha- Nuclear DNA content nisms of genome adaptation to different environmental conditions, among them climate and soil nutrients afecting Alstroemeria is the only Alstroemeriaceae genus for which plant productivity. The latitudinal gradient along which the DNA content (or C-value) has been estimated. Their spe- Chilean Alstroemeria species are distributed could be an cies are characterised by the presence of large genomes with interesting focus of study on these aspects (Borie and Rubio some C-values falling within the largest genome sizes of the 2003; Huygens et al. 2007; Stolpe and Undurraga 2016). Plantae kingdom (Sanso and Hunziker 1998), although they As mentioned above, karyotype modifcations in Alstro- are lower than the maximum 1C = 152.2 pg described within emeriaceae would have occurred through chromosome rear- monocots (Leitch and Leitch 2012). The correlation between rangements, some of them altering the centromere position C-values and total chromosome length (THL) has not been in the chromosomes as shown in the variation of MCA val- previously estimated for the family. The preliminary correla- ues among genera and among some species (Table 1, Fig. 3). tion made here between both variables was signifcant and However, DNA accumulation (e.g., transposable elements, positive (r = 0.92; p < 0.05) for the seven Alstroemeria spe- satellite DNA) appears to be involved in modifying the kary- cies studied so far, including Chilean, Brazilian and Argen- otype structure, accounting for the presence of high C-bands tinian taxa. It is interesting to note that fve Chilean species relative length and high DNA content in some species (Bui- (A. pelegrina, A. angustifolia, A. hookeri, A. aurea and A. tendijk and Ramanna 1996; Buitendijk et al. 1997, 1998). ligtu) with the largest genomes (large DNA C-values and Thus, some Alstroemeria species with high DNA content THL) are distributed to the south of latitude 33°S in Chile, show lower karyotype asymmetry (lower CVCL and MCA) whereas species distributed to the north of latitude 30°S (A. and higher chromosome size (and THL) (e.g. subspecies of philippi and A. magnifca) have smaller genomes (Buitendijk A. ligtu), while others (e.g., A. pelegrina, A. philippi, A. et al. 1997). DNA content is also correlated with the number magnifca, A. psittacina, A. inodora) with lower DNA con- of C-bands and the relative length of the C-bands within tent showed higher karyotype asymmetry (higher CVCL and the karyotypes (Table 5). The correlation between C-values MCA) and lower chromosome size (and THL). It is notewor- and geographical distribution has given rise to an interesting thy that species with intermediate asymmetry (intermediate investigative line in Alstroemeriaceae which has revealed CVCL and MCA) also have intermediate DNA C-values important aspects regarding genome modifcations because and chromosome size (and THL) (among them A. aurea, A. their species cover an extensive intercontinental distribu- hookeri, A. pulchra) (See Table 1 and Fig. 3). These obser- tion and inhabit diferent environments (e.g., arid and humid vations are consistent with our results regarding the signif- environments). The hypotheses proposed to date to explain cant and positive correlation between DNA C-values and the increase or reduction of the C-values within Alstroe- THL, and the signifcant and negative correlation between meria have alluded to the climate as an important factor DNA C-values and MCA and CVCL, respectively. DNA of pressure selection (e.g., rainfall regime and temperature) accumulation would tend to reduce size diferences between (Buitendijk and Ramanna 1996; Buitendijk et al. 1997; Han chromosomes of the karyotype in Alstroemeria, thus reduc- et al. 2000), as was also discussed for other plant groups ing the CVCL (from 55 in A. magnifca subsp. magnifca through quantile regression analysis (Knight and Ackerly to 21 in A. ligtu subsp. ligtu). Similarly, DNA addition 2002). Interestingly, within Liliaceae a similar tendency has decreases the variability in centromere position, reducing been described in some genera, with C-values related to hab- the MCA (from 52 in A. psittacina to 27 in A. ligtu subsp. itat and climate variation. Even certain morphological traits, ligtu). Considering reductions in both parameters (MCA and such as larger plant and perigon sizes, are associated with CVCL), more symmetrical karyotypes present higher DNA high DNA content, as was observed in Cardiocrinum and accumulation (e.g., subspecies of A. ligtu). This observation Lilium (Carta and Peruzzi 2016). In Alstroemeria, various suggests that the DNA is added evenly across the karyotype, DNA fractions have been characterised by means of cyto- resulting in uniform chromosomal shape and size. Never- molecular analyses (e.g., highly repetitive DNA sequences, theless, more species should be included in these analyses

1 3 Cytogenetics of Alstroemeriaceae Page 19 of 22 34 to prove their statistical signifcance more robustly. In the Human and animal rights This research did not involve any testing on case of Liliaceae, the tendency is reversed, since increases in humans or animals. DNA C-values have reduced karyotype asymmetry (CVCL) but increased the variability in centromere position, exhibit- ing high values of intrachromosomal asymmetry (Peruzzi References et al. 2009). Aagesen L, Sanso M (2003) The phylogeny of the Alstroemeriaceae, based on morphology, rps16 Intron, and rbcL sequence data. Syst Bot 28:47–69 Alzate F (2016) El género Bomarea (Alstroemeriaceae) en la Flora Conclusion de Colombia. (F. y N. Academia Colombiana de Ciencias Exactas, Ed.). Gente Nueva, Bogotá. https://​repos​itorio.​accef​ The integrated cytogenetic analysis presented here for yn.​org.​co/​handle/​001/​61 Alzate F, Mort M, Ramírez M (2008) Phylogenetic analyses of Alstroemeriaceae (based on 50 species across four gen- Bomarea (Alstroemeriaceae) based on combined analyses era—62 taxa) shows that the family has low chromosome of nrDNA ITS, psbA-trnH, rpoB-trnC and matK sequences. number diversity (2n = 16, 18, and 20). However, cytoge- Taxon 57:853–862. https://​doi.​org/​10.​1002/​tax.​573014 netic variations in karyotype morphology, asymmetry, total Arroyo S, Leuenberger B (1988b) A note on Luzuriaga marginata (Philesiaceae) from Paragonia. Herbertia 44:17–21 haploid length (27 species), and cytomolecular features (16 Arroyo S, Leuenberger B (1988a) morphology and taxonomic species) along the geographical range have been observed. history of Luzuriaga (Philesiaceae). Willdenowia 17:159–172 It is remarkable that South America concentrates the largest Arroyo-Leuenberger S (2001) Flora fanerogámica argentina Phile- cytogenetic diversity, which correlates with the greatest spe- siaceae, Asteliaceae y . Pro-Flora CONICET 72:1–7 cies diversity, as has been previously described in compari- Baeza C, Silva R (2016) Análisis citológico de una población de son to Oceania. However, only 24% of the family has been (L.) Mirb (Alstroemeriaceae) de la Región studied, with Alstroemeria being the genus with the highest del Biobío. Chile. Chilean J Agric Anim Sci 32:184–188. number of taxa analysed (27 taxa). This form of integrated https://​doi.​org/​10.​4067/​S0719-​38902​01600​50000​02 Baeza C, Schrader O, Ruiz E, Negritto M (2006) Análisis compara- analysis makes it possible to determine the most informa- tive del cariotipo en poblaciones de Alstroemeria ligtu subsp. tive cytogenetic features at the time of making decisions ligtu y A. ligtu subsp. simsii (Alstroemeriaceae) de Chile. for separating or grouping species together within genera Darwiniana 44:313–318. https://doi.​ org/​ 10.​ 14522/​ darwi​ niana.​ ​ and carry out subfamilial and intergeneric diferentiation on 2014.​442.​105 Baeza C, Schrader O, Budahn H (2007a) Characterization of geo- the basis of the presented analyses. Future research studies graphically isolated accessions in fve Alstroemeria L. spe- should include a higher number of species of the four genera cies (Chile) using FISH of tandemly repeated DNA sequences mainly on karyotype morphology, cytomolecular markers, and RAPD analysis. Pl Syst Evol 269:1–14. https://doi.​ org/​ 10.​ ​ and C-values. 1007/​s00606-​007-​0591-5 Information on Electronic Supplementary Mate- Baeza C, Schrader O, Ruiz E, Negritto M (2007b) Análisis com- parativo del cariotipo en poblaciones de Alstroemeria aurea R. rial Graham (Alstroemeriaceae) de Chile. Gayana Bot 64:33–39. https://​doi.​org/​10.​4067/​S0717-​66432​00700​01000​04 Online Resource 1. Alignment used to produce phylogeny. Baeza C, Alzate F, Negritto M, Ruiz E (2008a) El cariotipo de Bomarea patinii Baker subsp. patinii (Alstroemeriaceae). Gayana Bot 65:233–236. https://doi.​ org/​ 10.​ 4067/​ S0717-​ 66432​ ​ Supplementary Information The online version contains supplemen- 00800​02000​10 tary material available at https://doi.​ org/​ 10.​ 1007/​ s00606-​ 021-​ 01756-1​ . Baeza C, Schrader O, Ruiz E, Negritto M (2008b) Alstroemeria pres- liana Herb. (Alstroemeriaceae) in Chile from a cytogenetic Acknowledgements We would like to express our thanks to Núcleo perspective. Chilean J Agric Res 68:328–333. https://​doi.​org/​ de Estudios Ambientales (NEA), Dirección General de Investigación 10.​4067/​S0718-​58392​00800​04000​02 Alstroemeria diluta of the Universidad Católica de Temuco, and CONICYT PIA APOYO Baeza C, Ruiz E, Novoa P (2010a) Karyotype of CCTE AFB170008. Her. Bayer subsp. chrysantha (Alstroemeriaceae). Chilean J Agric Res 70:667–669 Funding Baeza C, Ruiz E, Negritto M (2010b) Comparative karyotypic anal- Environmental Sciences Nuclei and Research Direction, ysis in the Alstroemeria hookeri Lodd. (Alstroemeriaceae) Catholic University of Temuco, Chile; and CONICYT PIA APOYO complex sensu Bayer (1987). Genet Molec Biol 33:119–124. CCTE AFB170008. https://​doi.​org/​10.​1590/​S1415-​47572​01000​50000​12 Baeza C, Rojas G, Ruiz E (2011a) El cariotipo fundamental de Declarations Alstroemeria patagonica (Alstroemeriaceae). Bol Soc Argent Bot 46:313–315. https://​doi.​org/​10.​31055/​1851.​2372.​v55.​n4 Conflict of interest Authors declare that they have no confict of inter- Baeza C, Espejo J, Ruiz E (2011b) El cariotipo fundamental de Alstroemeria versicolor est. Ruiz et Pav. (Alstromeriaecae). Gay- ana Bot 68:327–329. https://​doi.​org/​10.​4067/​S0717-​66432​ 01100​02000​23

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