Complete genome sequence of a new begomovirus associated with yellow mosaic disease of Hemidesmus indicus in India

The complete DNA A genome of a virus isolate associated with yellow mosaic disease of a medicinal plant, Hemidesmus indicus, from India was cloned and sequenced. The length of DNA A was 2825 nucleotides, 35 nucleotides longer than the unit genome of monopartite begomoviruses. Comparison of the nucleotide sequence of DNA A of the virus isolate with those of other begomoviruses showed maximum sequence identity of 69 % to DNA A of ageratum yellow vein China virus (AYVCNV; AJ558120) and 68 % with tomato yellow leaf curl virus- LBa4 (TYLCV; EF185318), and it formed a distinct clade in phylogenetic analysis. The genome organization of the present virus isolate was found to be similar to that of Old World monopartite begomoviruses. The genome was considered to be monopartite, because association of DNA B and β satellite DNA components was not detected. Based on its sequence identity (<70 %) to all other begomoviruses known to date and ICTV (International Committee on Taxonomy of Viruses) species demarcating criteria (<89 % identity), it is considered a member of a novel begomovirus species, and the tentative name “Hemidesmus yellow mosaic virus” (HeYMV) is proposed.     

A new, previously undescribed monopartite begomovirus infecting Premna serratifolia in Vietnam

The complete genome sequence of a monopartite begomovirus isolate infecting Creek Premna (Premna serratifolia L.) plants that exhibited leaf curl, vein swelling, and enation symptoms in Nha Trang, Vietnam, was cloned and sequenced. It comprises 2,753 nucleotides (JQ793786) and has a typical organization of begomoviruses DNA-A with AV1 and AV2 open reading frames (ORFs) in the viral-sense strand and AC1, AC2, AC3, AC4 and AC5 ORFs in the complementary-sense strand. The full-length genome sequence of the isolate (clone VN7) shared the highest level of nucleotide sequence identity (83 %) with the isolate IN:Pusa:Tb:10 of tobacco leaf curl Pusa virus (HQ180391). The phylogenetic relationship of VN7 to other begomoviruses was also investigated. VN7 grouped most closely with a clade containing begomoviruses from China, India and Japan. According to the current taxonomic criteria for the genus Begomovirus, family Geminiviridae, the isolate VN7 represents a new species, herein named “Premna leaf curl virus” (PrLCV).     

Complete nucleotide sequence of a monopartite Begomovirus and associated satellites infecting Carica papaya in Nepal

Carica papaya (papaya) is a fruit crop that is cultivated mostly in kitchen gardens throughout Nepal. Leaf samples of C. papaya plants with leaf curling, vein darkening, vein thickening, and a reduction in leaf size were collected from a garden in Darai village, Rampur, Nepal in 2010. Full-length clones of a monopartite Begomovirus, a betasatellite and an alphasatellite were isolated. The complete nucleotide sequence of the Begomovirus showed the arrangement of genes typical of Old World begomoviruses with the highest nucleotide sequence identity (>99 %) to an isolate of Ageratum yellow vein virus (AYVV), confirming it as an isolate of AYVV. The complete nucleotide sequence of betasatellite showed greater than 89 % nucleotide sequence identity to an isolate of Tomato leaf curl Java betasatellite originating from Indonesian. The sequence of the alphasatellite displayed 92 % nucleotide sequence identity to Sida yellow vein China alphasatellite. This is the first identification of these components in Nepal and the first time they have been identified in papaya.     

Molecular characterization of a distinct bipartite begomovirus species infecting tomato in India

A distinct bipartite begomovirus was found associated with tomato plants showing yellowing, curling, and crumpling of the leaves, in a sub-temperate region in India. The complete DNA-A and DNA-B components were amplified through rolling circle amplification (RCA) using Φ-29 DNA polymerase and characterized. The DNA-A of the isolate was comprised of 2,756 nucleotides, encoding six open reading frames (ORFs) and DNA-B that of 2,725 nucleotides, encoding two ORFs. Genome organization of the isolate was typical of an old world bipartite begomovirus. Comparisons showed that DNA-A and its intergenic region (IR) have the highest sequence identity (86% and 84%, respectively) with the Tomato leaf curl New Delhi virus (ToLCNDV; DQ116885) and some other begomoviruses (>84%) reported from cucurbits and tomato. This data suggested that the isolate is a distinct begomovirus species for which a name Tomato leaf curl Palampur virus (ToLCPMV) is proposed. DNA-B showed the maximum sequence identity (73%) with Tomato leaf curl New Delhi virus-India-[Pakistan:Dargai:T5/6:2001] (AY150305). The common region (CR) of DNA-A and DNA-B showed 94% sequence similarity with each other. In the present study, phylogenetic relationship of this new species was also established with different begomoviruses reported from tomato and other begomoviruses showing highest homologies with complete DNA-A and DNA-B sequences. ToLCPMV is being reported from a sub-temperate region in India which was previously unaffected by begomoviruses and its whitefly vector. An erratum to this article can be found at     

A new monopartite begomovirus isolated from <Emphasis Type="Italic">Hibiscus cannabinus</Emphasis> L. in India

Summary Yellow vein mosaic disease of mesta is whitefly-transmitted and found in endemic form in different parts of India, causing great economic losses. Full-length DNA-A of a begomovirus infecting mesta was cloned and sequenced. The genome was homologous to the DNA-A of monopartite begomoviruses originating from the Old World, with six conserved open reading frames. The complete nucleotide sequence of the DNA-A molecule of the present isolate was 2728 nucleotides in length, having the highest sequence identity (83.5%) with an Indian begomovirus causing cotton leaf curl disease. It thus belongs to a novel geminivirus species, and the name Mesta yellow vein mosaic virus is proposed.     

Molecular characterization of a distinct begomovirus infecting <Emphasis Type="Italic">Allamanda cathartica</Emphasis> in Guangdong,China

Virus isolate G10 was obtained from diseased allamanda plants showing leaf curl symptoms in Guangdong, China. The full-length nucleotide sequence of a DNA-A-like molecule of G10 was cloned and sequenced; it comprises 2755 nucleotides and has a typical begomovirus genome organization with six conserved open reading frames. When compared with the DNA-A sequences of other begomoviruses, the complete nucleotide sequence of DNA-A of G10 had the highest sequence identity (81.2%) to tomato leaf curl Guangdong virus (ToLCGuV) isolate G2. This is less than the 89% identity in the complete genome that has been defined as the threshold value for demarcation of species in the genus Begomovirus. The molecular data show that isolate G10 from allamanda in Guangdong, China is a distinct Begomovirus species, for which the name Allamanda leaf curl virus (AlLCV) is proposed. The GenBank accession number of the sequence reported in this paper is EF602306.     

Molecular characterization of distinct bipartite begomovirus infecting bhendi (Abelmoschus esculentus L.) in India

Yellow vein mosaic disease of okra is a whitefly transmitted begomovirus causing heavy economic loss in different parts of India. The okra isolate (OY131) of this virus from a bhendi plant [(Abelmoschus esculentus L.) Moench] showing yellow vein mosaic, vein twisting, reduced leaves, and a bushy appearance in the Palem region, New Delhi, India, was characterized in the present study. The complete DNA-A and DNA-B sequences have been determined and are comprised of 2,746 and 2,703 nucleotides, respectively. The betasatellite (DNA-β) component was absent in the sample. The genome organization was typically of biparite begomoviruses, which were characterized earlier. Comparison of DNA-A component with other known begomoviruses suggest that this virus, being only distantly related (<85.9% similarity with its nearest relative, BYVMV) to other known begomoviruses, is a new species. We have tentatively assigned the genome to a novel geminivirus species Bhendi yellow vein mosaic Delhi virus [BYVDV-IN (India: Delhi: okra)]. DNA-B showed highest sequence identity (87.8% identical) to that of a ToLCNDV (AY158080). The phylogenetic analysis of the present isolate is distinct from all other viruses; however clusters with ToLCNDV group infect different crops. The recombination analysis revealed that this isolate has sequences originated from ToLCNDV. This is the first known bhendi yellow vein mosaic disease associated bipartite begomovirus from India.     

Interaction of tomato yellow leaf curl virus with diverse betasatellites enhances symptom severity

The complete nucleotide sequence was determined for a begomovirus isolated from tomato exhibiting leaf curling and yellowing symptoms in Tochigi Prefecture in Japan. The genome organization of this virus was similar to those of other Old World monopartite begomoviruses. Neither a DNA betasatellite nor a DNA-B component was detected. It had the highest total nucleotide sequence identity (99%) with tomato yellow leaf curl virus-Israel[Japan:Tosa:2005] (TYLCV-IL[JR:Tos:05]) and TYLCV-Israel[Japan:Haruno:2005] (TYLCV-IL[JR:Han:05]). Its coat protein V1 also showed an identical amino acid sequence with those of TYLCV-IL[JR:Tos:05] and TYLCV-IL[JR:Han:05]. Thus, the begomovirus was determined to be an isolate of TYLCV-IL designated as TYLCV-Israel[Japan:Tochigi:2007] (TYLCV-IL[JR:Toc:07]). We investigated the interaction of TYLCV-IL[JR:Toc:07] with two known satellites associated with tomato yellow dwarf disease in Japan, tobacco leaf curl Japan betasatellite [Japan:Ibaraki:2006] and honeysuckle yellow vein mosaic betasatellite [Japan:Nara:2006], as well as with tomato leaf curl Philippines betasatellite [Philippines:Laguna1:2008], in tomato and Nicotiana benthamiana plants. TYLCV-IL[JR:Toc:07] trans-replicated these betasatellites, inducing more severe tomato yellow leaf curl disease-related symptoms than TYLCV-IL[JR:Toc:07] alone.     

A betasatellite-dependent begomovirus infects ornamental rose: characterization of begomovirus infecting rose in Pakistan

The Begomovirus genus of the family Geminiviridae comprises the largest group of geminiviruses. The list of begomoviruses is continuously increasing as a result of improvement in the methods for identification. Ornamental rose plants (Rosa chinensis) with highly stunted growth and leaf curling were found in Faisalabad, Pakistan. Plants were analyzed for begomovirus infection, through rolling circle amplification and PCR methods. Based on complete genome sequence homologies with other begomoviruses, a new begomovirus species infecting the rose plants was discovered. In this paper, we propose a new species name, Rose leaf curl virus (RoLCuV), for the virus. RoLCuV showed close identity (83 %) with Tomato leaf curl Pakistan virus, while associated betasatellite showed 96 % identity with Digera arvensis yellow vein betasatellite (DiAYVB), justifying a new isolate for the betasatellite. Recombination analysis of newly identified begomovirus revealed it as a recombinant of tomato leaf curl Pakistan virus from its coat protein region. The infectious molecules for virus/satellite were prepared and inoculated through Agrobacterium tumefaciens to N. benthamiana plants. RoLCuV alone was unable to induce any level of symptoms on N. benthamiana plants, but co-inoculation with cognate betasatellite produced infection symptoms. Further investigation to understand the trans-replication ability of betasatellites revealed their flexibility to interact with Rose leaf curl virus.     

Complete nucleotide sequence of a begomovirus and associated betasatellite infecting croton (Croton bonplandianus) in Pakistan

The complete sequences of a begomovirus and an associated betasatellite isolated from Croton bonplandianus originating from Pakistan were determined. The sequence of the begomovirus showed the highest level of nucleotide sequence identity (88.9%) to an isolate of papaya leaf curl virus and thus represents a new species, for which we propose the name Croton yellow vein virus (CYVV). The sequence of the betasatellite showed the highest levels of sequence identity (82 to 98.4%) to six sequences in the databases that have yet to be reported, followed by isolates of tomato leaf curl Joydebpur betasatellite (48.7 to 52.5%). This indicates that the betasatellite identified here (and the six sequences in the databases) is an isolate of a newly identified species for which the name Croton yellow vein mosaic betasatellite (CroYVMB) is proposed. For the begomovirus, an analysis of the sequence indicates that it has a recombinant origin.     

Characterization of a new begomovirus and a beta satellite associated with the leaf curl disease of French bean in northern India

Begomoviruses are emerging as serious threat to many crops throughout the world particularly in tropical and sub-tropical regions. A leaf curl disease with symptoms typical of infection by many begomoviruses was observed in French bean (Phaseolus vulgaris) at Kanpur, India, during 2010–2012. The disease caused downward leaf curling and made the plants unproductive. The disease was transmitted from infected to healthy plants through whitefly (Bemisia tabaci). The products of five samples digested with EcoRI yielded DNA fragments of about 2.7 kb. The complete sequence of the Fb1 sample comprised 2,741 nucleotides with genome organization typical of begomoviruses having two ORFs in virion-sense and five ORFs in complementary-sense separated by an intergenic region with begomovirus conserved nonanucleotide sequence, TAATATTAC. The complete DNA-A sequence homology was most closely related to Cotton leaf curl Bangalore virus with 80 % nucleotide sequence identity. Based on the demarcation criteria for identifying a begomovirus species, Fb1 is considered as a distinct begomovirus species, named French bean leaf curl virus and designated as FbLCV-[IN:Knp:12]. The complete sequence of associated satellite DNA-β comprises 1,379 nucleotides with single ORF and has 80 % identity with Papaya leaf curl beta satellite. There was no evidence of recombination in DNA-A of FbLCV and associated beta satellite DNA molecule.     

Molecular characterization of a new begomovirus infecting Senecio scandens in China

Three begomovirus isolates, G46, G83 and G84 from Senecio scandens showing yellow mosaic symptoms were collected from Guangxi Province, P.R. China. The isolates were detected by PCR using universal primers for begomoviruses. Comparison of partial DNA-A sequences (approximately 500 bp) of the isolates revealed their 98.7-99.1% identity. The isolate G46, chosen for complete DNA-A sequencing, consisted of 2746 nt and had a typical genomic organization of begomoviruses. The G46 DNA-A had the highest sequence identity (72.4%) with that of Ageratum leaf curl virus among begomoviruses. The molecular data suggest that the isolate G46 is a new begomovirus (species), for which the name Senecio yellow mosaic virus (Senecio yellow mosaic virus) is proposed.     

The begomoviruses Honeysuckle yellow vein mosaic virus and Tobacco leaf curl Japan virus with DNAbeta satellites cause yellow dwarf disease of tomato

The complete nucleotide sequences of two begomoviruses (Nara virus-1 and Nara virus-2), a satellite DNA (DNAbeta-Nara) and defective DNAs were obtained from honeysuckle (Lonicera japonica) showing characteristic yellow vein mosaic symptoms in Nara Prefecture, Japan. One begomovirus (Ibaraki virus) and a satellite DNA (DNAbeta-Ibaraki) was isolated and cloned from honeysuckle plants exhibited typical yellowing of veins and small elliptical shaped enations along veins on the under side of the leaves in Ibaraki Prefecture, Japan. The genome organization of the three viruses is the same as those of other Old World monopartite begomoviruses. Nara virus-1 had overall nucleotide sequence identity with Nara virus-2 of 94% and Ibaraki virus of 90%. DNAbeta-Nara had overall nucleotide sequence identity with DNAbeta-Ibaraki of 83%. Comparison of the nucleotide sequences with other begomoviruses revealed that Nara virus-1 and Nara virus-2 are strains of Honeysuckle yellow vein mosaic virus (HYVMV), hence named as HYVMV-Nara1 and HYVMV-Nara2, whereas Ibaraki virus was a strain of Tobacco leaf curl Japan virus (TbLCJV), designated as TbLCJV-Hs[Iba]. HYVMV-Nara1 and HYVMV-Nara2 have hybrid genomes, which are likely to have formed recombination between HYVMV and TbLCJV. TbLCJV-Hs[Iba] or HYVMV-Nara2 could infect and cause yellowing, leaf crinkling and stunting symptoms when partial tandem dimeric constructs were agroinoculated on tomato plants. However, in the presence of DNAbeta, both TbLCJV-Hs[Iba] or HYVMV-Nara2 produced more severe stunting symptoms in tomato plants. Therefore, these viruses along with their satellites are causal agents of tomato yellow dwarf disease in Japan, and honeysuckle acts as a potential reservoir host. Previously available evidence indicated that DNAbeta elements do not contain iteron sequences of their helper viruses; hence this is the first evidence that DNAbeta satellites have the iteron of their helper virus.     

Identification and molecular characterization of two begomoviruses from Pouzolzia zeylanica (L.) Benn. exhibiting yellow mosaic symptoms in adjacent regions of China and Vietnam

Two monopartite begomoviruses were isolated from Pouzolzia zeylanica (L.) Benn. plants showing yellow mosaic symptoms in Gaoyao, Guangdong Province, China (GD1) and in Phu Tho, Vietnam (VN), respectively. A comparison of the complete genome sequence of GD1 (2,739 nucleotides [nt]) with VN (2,741 nt) indicated that they shared 86.2 % nt sequence identity. GD1 and VN shared the highest nucleotide sequence identity at 86.7 % and 91.4 % respectively, with isolate TY01 of pouzolzia golden mosaic virus (PGMV-TY01), another begomovirus isolated from P. zeylanica. Phylogenetic analysis revealed that GD1, VN, and PGMV-TY01 were members of a distinct begomovirus clade. Based on the ICTV guidelines for begomoviral species demarcation, GD1 belongs to a new begomovirus species, for which the name Pouzolzia yellow mosaic virus is proposed. Likewise, VN represents a previously unreported strain of PGMV. Recombination analysis predicted that VN was a recombinant between PGMV-TY01 and ageratum yellow vein China virus isolate G13 (AYVCNV-G13), and that PGMV-TY01 and VN were likely the parents of GD1 through recombination with allamanda leaf curl virus isolate G10 (AlLCV-G10), a begomovirus endemic to Guangdong Province of China.     

Siegesbeckia yellow vein virus is a distinct begomovirus associated with a satellite DNA molecule

Summary Leaf samples of Siegesbeckia glabrescens showing yellow vein, enation, and stunting symptoms were collected in Guangdong province, China. A specific 500-bp product was consistently detected in total DNA extracts, amplified with universal primers specific for members of the genus Begomovirus. Comparison of partial DNA sequences revealed that these virus isolates were identical, and therefore isolates GD13, GD24 and GD27 were selected for further sequence analysis. The complete nucleotide sequences of GD13, GD24 and GD27 were all found to be 2768 nucleotides (nts) long, with two open reading frames (ORFs) in the virion-sense strand and four ORFs in the complementary-sense strand, typical of the Old World begomoviruses. Sequence identities among the three isolates ranged from 99.7 to 99.8%. When compared with other reported sequences of begomoviruses, GD13 was most closely related to papaya leaf curl China virus (AJ876548), with a sequence identity of 76.8%. In addition, all isolates were found to be associated with DNAβ molecules. The complete DNAβ sequences of isolates GD13, GD24 and GD27 were determined. Sequence analysis showed that they had highest sequence identity with DNAβ of Eupatorium yellow vein virus (AJ438938) (44.0, 43.9 and 45.6% identity). GD13, GD24 and GD27 are considered to be isolates of a distinct begomovirus species for which the name Siegesbeckia yellow vein virus (SgYVV) is proposed.     

Characterization of begomovirus components from a weed suggests that begomoviruses may associate with multiple distinct DNA satellites

A begomovirus disease complex associated with Sonchus arvensis, a common weed in Pakistan was studied using cloning, nucleic acid sequencing and phylogenetic analysis. The complex associated with this weed consists of a monopartite begomovirus and several distinct betasatellites and alphasatellites. The monopartite begomovirus associated with yellow vein disease of Sonchus arvensis showed 95–99% nucleotide sequence identity with Alternanthera yellow vein virus (AlYVV) reported from China, Vietnam and India. Two betasatellites were isolated from S. arvensis: one sharing between 91.4 and 95.3% nucleotide sequence identity with isolates of Ageratum yellow leaf curl betasatellite (AYLCB), and the other sharing between 78.2 and 99.9% identity with isolates of Cotton leaf curl Multan betasatellite (CLCuMB). Two alphasatellites were identified: one was homologous to Potato leaf curl alphasatellite (PotLCuA), while the other was closely related to Hibiscus leaf curl alphasatellite (HLCuA). Thus, AlYVV in S. arvensis is associated with satellites shown previously to be associated with other begomoviruses in Pakistan. Our results suggest that monopartite begomoviruses may associate with distinct satellites that are prevalent in the region.     

Molecular characterization of Chilli leaf curl virus and satellite molecules associated with leaf curl disease of Amaranthus spp

Amaranthus, collectively known as amaranth, is an annual or short-lived perennial plant used as leafy vegetables, cereals and for ornamental purposes in many countries including India. During 2011, leaf samples of Amaranthus plants displaying leaf curling, leaf distortion, leaf crinkling and yellow leaf margins were collected from Banswara district, Rajasthan in India. Full-length clones of a monopartite begomovirus, a betasatellite and an alphasatellite were characterized. The complete nucleotide sequence of the isolated begomovirus features as a typical ‘Old World’ begomovirus with the highest nucleotide per cent identity with Chilli leaf curl virus and hence, considered as an isolate of Chilli leaf curl virus. The complete nucleotide sequences of betasatellite and alphasatellite possess maximum nucleotide identity with Tomato yellow leaf curl Thailand betasatellite and Chilli leaf curl alphasatellite, respectively. This is the first report of the association of chilli-infecting begomovirus and satellite molecules infecting a new host, Amaranthus, causing leaf curl disease.     

Role of betasatellite in the pathogenesis of a bipartite begomovirus affecting tomato in India

Betasatellites are commonly associated with tomato leaf curl disease caused by begomoviruses in India. This study demonstrates the role of a betasatellite in the pathogenesis of tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus affecting tomato in India. For infection, accumulation, systemic movement and disease induction by ToLCNDV, co-infection by the associated betasatellite was not essential, as the DNA A alone of ToLCNDV could infect tomato and Nicotiana benthamiana and induce mild symptoms, but DNA B or Cotton leaf curl Multan betasatellite (CLCuMB) was required for development of typical leaf curl symptoms. The symptoms were most severe in plants infected with all three components, indicating a role of the betasatellite in the pathogenesis of ToLCNDV. The plants infected with ToLCNDV DNA A alone had limited accumulation of viral DNA, which increased by many times in plants co-infected with DNA B or/and betasatellite. However, the plants infected with all three components accumulated 20 times less betasatellite DNA than the plants infected with DNA A and betasatellite. The increase in the amount of viral DNAs was also reflected in the commensurate increase in symptom severity and transmissibility by whitefly, Bemisia tabaci.     

Identification and molecular characterization of begomovirus and associated satellite DNA molecules infecting <Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>

Monopartite begomoviruses comprise DNA-A as the main genome and associated satellite DNAs. Viral DNA extracted from guar (Cyamopsis tetragonoloba) showing leaf curl symptoms exhibited positive amplification of coat protein (CP) gene of DNA-A component, suggesting the presence of begomovirus. Full length DNA-A was amplified by primer pair re-designed from CP gene nucleotide sequence. The associated alphasatellite and betasatellite DNA molecules were amplified and sequenced, confirming the presence of monopartite begomovirus. Sequence comparisons showed 89% identity with other begomoviruses. The Neighbor-Joining tree based on full length DNA-A nucleotide sequence showed that the guar infecting begomovirus clustered separately from other known begomoviruses. The betasatellite shared a high (96%) nucleotide identity to Cotton leaf curl Multan betasatellites. The alphasatellite showed 91% nucleotide identity to alphasatellite associated with begomovirus infecting Okra. Recombination analyses showed three recombinant fragments in DNA-A, two in betasatellite, and four in alphasatellite. The results suggest that the begomovirus identified in this study was a new recombinant virus. Its name was proposed as Cyamopsis tetragonoloba leaf curl virus (CyTLCuV).     

Begomoviruses infecting weeds in Cuba: increased host range and a novel virus infecting <Emphasis Type="Italic">Sida rhombifolia</Emphasis>

As a result of surveys conducted during the last few years to search for wild reservoirs of begomoviruses in Cuba, we detected a novel bipartite begomovirus, sida yellow mottle virus (SiYMoV), infecting Sida rhombifolia plants. The complete genome sequence was obtained, showing that DNA-A was 2622 nucleotides (nt) in length and that it was most closely related (87.6% nucleotide identity) to DNA-A of an isolate of sida golden mosaic virus (SiGMV) that infects snap beans (Phaseolus vulgaris) in Florida. The DNA-B sequence was 2600 nt in length and shared the highest nucleotide identity (75.1%) with corchorus yellow spot virus (CoYSV). Phylogenetic relationship analysis showed that both DNA components of SiYMoV were grouped in the Abutilon clade, along with begomoviruses from Florida and the Caribbean islands. We also present here the complete nucleotide sequence of a novel strain of sida yellow vein virus found infecting Malvastrum coromandelianum and an isolate of euphorbia mosaic virus that was found for the first time infecting Euphorbia heterophylla in Cuba.